CHIMERIC AND HUMANIZED ANTIBODIES TO alpha5beta1 INTEGRIN THAT MODULATE ANGIOGENESIS

ABSTRACT

The present invention provides chimeric and humanized antibodies that specifically recognize α5β1 integrin, and methods for using the antibodies for reducing or inhibiting angiogenesis in a tissue. Also provided are methods of determining therapeutically acceptable doses of the antibodies and pharmaceutical compositions including the same.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/830,956, filed Apr. 23, 2004, which is a continuation-in-part of U.S.application Ser. No. 10/724,274, filed Nov. 26, 2003 which, claimspriority from U.S. Provisional Application No. 60/429,743, filed Nov.26, 2002 and U.S. Provisional Application 60/508,149, filed Sep. 30,2003, each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention provides chimeric and humanized antibodies thatspecifically recognize α5β1 integrin, and methods for using theantibodies for reducing or inhibiting angiogenesis in a tissue. Alsoprovided are methods of determining therapeutically acceptable doses ofthe antibodies and pharmaceutical compositions including the same.

BACKGROUND OF THE INVENTION

Angiogenesis is the process whereby new blood vessels are formed.Angiogenesis, also called neovascularization, occurs normally duringembryogenesis and development, and occurs in fully developed organismsduring wound healing and placental development. In addition,angiogenesis occurs in various pathological conditions including: oculardiseases such as diabetic retinopathy and macular degeneration due toneovascularization; conditions associated with tissue inflammation suchas rheumatoid arthritis and inflammatory bowel disease; and cancer,where blood vessel formation in the growing tumor provides oxygen andnutrients to the tumor cells, as well as providing a route via whichtumor cells metastasize throughout the body. Since millions of peoplearound the world are afflicted by these diseases, a considerable efforthas been made to understand the mechanisms involved in angiogenesis inorder to develop methods for detecting and inhibiting such undesirableangiogenesis.

Angiogenesis occurs in response to stimulation by one or more knowngrowth factors, and also may involve other as yet unidentified factors.Endothelial cells, which are the cells that line mature blood vessels,normally do not proliferate. However, in response to an appropriatestimulus, the endothelial cells become activated and begin toproliferate and migrate into unvascularized tissue to form new bloodvessels. In some cases, precursor cells are activated to differentiateinto endothelial cells, which form new blood vessels.

Blood vessels are surrounded by an extracellular matrix. In addition tostimulation by growth factors, angiogenesis depends on interaction ofthe endothelial cells with the extracellular matrix, as well as witheach other. The activation of endothelial cells by growth factors andthe migration into and interaction with the extracellular matrix andwith each other is dependent on cell surface receptors expressed by theendothelial cells. These cell surface receptors, which include growthfactor receptors and integrins, interact specifically with particularmolecules.

In pathological conditions such as age-related macular degeneration anddiabetic retinopathy, decreased availability of oxygen to the retinaresults in a hypoxic condition that stimulates the secretion ofangiogenic growth factors such as vascular endothelial growth factors(VEGF). This secretion induces abnormal migration and proliferation ofendothelial cells into tissues of the eye. This results invascularization of ocular tissues and can induce corneal scarring,retinal detachment and fluid accumulation in the choroid, each of whichcan adversely affect vision and lead to blindness.

Angiogenesis also is associated with the progression and exacerbation ofinflammatory diseases, including psoriasis, rheumatoid arthritis,osteoarthritis, and inflammatory bowel diseases such as ulcerativecolitis and Crohn's disease. In inflammatory arthritic disease, forexample, influx of lymphocytes into the region surrounding the jointsstimulates angiogenesis in the synovial lining. This increasedvasculature provides a means for greater influx of leukocytes, whichfacilitates the destruction of cartilage and bone in the joint.Angiogenic vascularization that occurs in inflammatory bowel diseaseresults in similar effects in the bowel.

The growth of capillaries into atherosclerotic plaques in the coronaryarteries represents another pathological condition associated withgrowth factor induced angiogenesis. Excessive blood flow intoneovascularized plaques can result in rupture and hemorrhage of theblood-filled plaques, releasing blood clots that can result in coronarythrombosis.

The involvement of angiogenesis in such diverse diseases as cancer,ocular disease and inflammatory diseases has led to an effort toidentify methods for specifically inhibiting angiogenesis as a means totreat these diseases. For cancer patients, such methods of treatment canprovide a substantial advantage over currently used methods such aschemotherapy, which kill or impair not only the target tumor cells butalso normal proliferating cells in the patient, such as blood cells,epithelial cells, and cells lining the intestinal lumen. Suchnon-specific killing by chemotherapeutic agents results in side effectsthat are, at best, unpleasant, and can often result in unacceptablepatient morbidity, or mortality. In fact, the undesirable side effectsassociated with cancer therapies often limit the treatment a patient canreceive.

BRIEF SUMMARY OF THE INVENTION

The present invention provides therapeutic chimeric and humanizedantibodies directed against α5β1 integrin; methods for purification ofthese antibodies, and methods for their use in treating conditionscomprising undesirable tissue angiogenesis.

In one embodiment the invention includes a nucleic acid encoding apolypeptide of a chimeric or humanized anti-α5β1 integrin antibody,having 65%, preferably more than 75%, more preferably 85%, 90%, 95%, 97%or 99% sequence identity to one or more of the amino acid sequencesselected from the group consisting of SEQ ID NOS: 1-12, 16, 18, 20, 22,25-26, 28, 31-32. Most preferably, the nucleic acid encodes apolypeptide of a chimeric or humanized anti-α5β1 integrin antibodycomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 2-6, 8-12, 16, 18, 20, 22, 25-26, 28, 31-32. The peptideencoded by this nucleic acid can be a single-chain antibody or Fab, inaddition to a Fab or antibody comprising several peptides bound bydisulfide bridges.

The invention also includes a polypeptide having 65%, preferably morethan 75%, more preferably 85%, 90%, 95%, 97% or 99% sequence identity toone or more of the amino acid sequences selected from the groupconsisting of SEQ ID NOS: 1-12, 16, 18, 20, 22, 25-26, 28, 31-32. Mostpreferably, the nucleic acid encodes a polypeptide comprising one ormore of the amino acid sequences selected from the group consisting ofSEQ ID NOS: 2-6, 8-12, 16, 18, 20, 22, 25-26, 28, 31-32. These peptidesinclude chimeric, human and humanized antibodies and Fab fragments.

In another embodiment the invention includes chimeric anti-α5β1 integrinantibodies. These antibodies comprise a first polypeptide from a firstsource comprising an amino acid sequence having a sequence 65%,preferably more than 75%, more preferably 85%, 90%, 95%, 97% or 99%identical to an amino acid sequence selected from the group consistingof SEQ ID NOS: 1, 7, 16, 18, 20, 22; and a second polypeptide from asecond source having a sequence 65%, preferably more than 75%, morepreferably 85%, 90%, 95%, 97% or 99% identical to a constant region ofan antibody of the second source wherein the first and secondpolypeptides form a protein complex that is immunoreactive with α5β1integrin. In a preferred embodiment the second source of the constantregion is a human IgG. In another preferred embodiment, the constantregion is a human IgG4.

In another preferred embodiment, the chimeric antibodies comprise afirst polypeptide sequence from a first source comprising one or moreamino acid sequences selected from the group consisting of SEQ ID NOS:1, 7, 16, 18, 20, 22; and a second polypeptide sequence from a secondsource comprising a constant region sequence of an antibody of thesecond source wherein the first and second polypeptide sequences form aprotein complex that is immunoreactive with α5β1 integrin.

In a most preferred embodiment, the invention includes a chimericanti-α5β1 integrin antibody comprising the heavy chain amino acidsequence SEQ ID NO: 25 and the light chain amino acid sequence SEQ IDNO: 26.

In an alternative embodiment, the invention includes a nucleic acidencoding a chimeric anti-α5β1 integrin antibody heavy chain variableregion comprising SEQ ID NO: 19, and a nucleic acid encoding a chimericanti-α5β1 integrin antibody heavy chain variable region comprising SEQID NO: 21.

In a further preferred embodiment, the invention includes a Fab fragmentderived from the chimeric anti-α5β1 integrin antibody comprising heavychain amino acid sequence SEQ ID NO: 25 and light chain amino acidsequence SEQ ID NO: 26. In a most preferred embodiment, the Fab fragmentcomprises the heavy chain amino acid sequence SEQ ID NO: 28 and thelight chain amino acid sequence SEQ ID NO: 26.

In a further preferred embodiment, the invention includes a humanizedantibody derived from the chimeric anti-α5β1 integrin antibodycomprising heavy chain amino acid sequence SEQ ID NO: 25 and light chainamino acid sequence SEQ ID NO: 26. In a most preferred embodiment, thehumanized antibody comprises the heavy chain amino acid sequence SEQ IDNO: 28 and the light chain amino acid sequence SEQ ID NO: 26.

In another embodiment, the invention includes an expression vectorcomprising the any one or more of the nucleic acids selected from thegroup consisting of SEQ ID NOS: 15, 17, 19, 21, 23, 24, 27, 29, and 30.In a preferred embodiment the expression vector comprises SEQ ID NOS: 19and 21.

In another embodiment, the invention includes a cell transformed by anexpression vector comprising the any one or more of the nucleic acidsselected from the group consisting of SEQ ID NOS: 15, 17, 19, 21, 23,24, 27, 29, and 30. In a preferred embodiment the expression vectorcomprises SEQ ID NOS: 19 and 21.

In another embodiment the invention includes pharmaceutical compositionscomprising the chimeric or humanized anti-α5β1 integrin antibodiesdescribed herein. In some embodiments, these compositions may containagents that enhance the uptake or localization of the therapeuticcomponent, decrease inflammation, or otherwise provide localized relief.

In one aspect of this embodiment, the pharmaceutical compositioncomprises a topical cream that is applied directly to the injuredtissue. In another aspect, the pharmaceutical is an eye drop solutionthat is applied directly to the injured eye. In still another aspect isa pharmaceutical that is an injectable that can be applied systemicallyto treat injured tissue in one or both eyes of an individual or toinhibit neoangiogenesis in tumor tissue.

In another embodiment the invention includes methods of controllingvascularization in injured tissue. These methods comprise applying oneor more doses of a chimeric or humanized anti-α5β1 integrin antibody tothe injured tissue, where the injury to the tissue can be the result ofphysical or chemical damage, or disease.

In another embodiment, the invention includes a method of treatment ofangiogenesis associated ocular diseases comprising applying one or moredoses of a chimeric or humanized anti-α5β1 integrin antibody to theinjured tissue. In one embodiment of the method, the humanized anti-α5β1integrin antibody exhibits significant inhibition of subretinalhemorrhage due to a VEGF/bFGF Hydron™ Implant pellet (hydrogel polymer)in rabbit eye. In some embodiments, the chimeric or humanized anti-α5β1integrin antibody comprises amino acids selected from the groupconsisting of SEQ ID NOS: 20, 22, 25, 26, 28, 31 and 32, and in apreferred embodiment of the method, the antibody is selected from M200or F200. In some embodiments, the ocular diseases are selected from thegroup consisting of macular degeneration, diabetic retinopathy, andchoroidal neovascularization. In some embodiments, the method ofapplying the antibody comprises intravitreal injection. In otherembodiments, intravenous injection may be used.

In another embodiment, the invention includes a method of treatment ofgrowth factor associated ocular diseases comprising applying one or moredoses of a chimeric or humanized growth factor neutralizing antibody tothe injured eye tissue. In one embodiment, the method may be usedwherein the growth factor is VEGF. In one embodiment of the method, thehumanized anti-α5β1 integrin antibody exhibits significant inhibition ofsubretinal hemorrhage due to a VEGF/bFGF Hydron™ Implant pellet(hydrogel polymer) in rabbit eye. In preferred embodiments, the methodinvolves applying a chimeric or humanized growth factor neutralizingantibody that is an anti-α5β1 integrin antibody comprising amino acidsselected from the group consisting of SEQ ID NOS: 20, 22, 25, 26, 28, 31and 32. In one particularly preferred embodiment, the chimeric orhumanized growth factor neutralizing antibody is M200 or F200.

In another embodiment the invention includes a method of administering atherapeutic antibody comprising: providing a pharmaceutical including atherapeutic antibody comprising a variable heavy chain region having asequence selected from the group consisting of SEQ ID NOS: 2-6, 16, 20and a variable light chain region independently selected from the groupconsisting of SEQ ID NOS: 8-12, 18, 22; and applying the therapeuticantibody to an injured tissue. In this embodiment of the invention, theinjured tissue responds to injury by increasing its blood flow throughneovascularization and the therapeutic antibody inhibits thisneovascularization. In one aspect the method involves injectingtherapeutic antibodies intravitreally into a diseased or injured eye ofan individual who has two afflicted eyes; intravitreal injection of oneeye being sufficient to treat both eyes.

In another embodiment the invention includes a process for thepurification of anti-α5β1 integrin antibodies. The method comprisesabsorbing the antibody onto an antibody affinity matrix bound to asubstrate and eluting the antibody from the substrate-bound antibodyaffinity matrix using an eluting solution having a pH of about 3.0 toabout 5.5. The process may further comprise recovering the purifiedantibody. Antibodies amenable to purification using this procedureinclude those comprising at least two CDR regions selected independentlyfrom those present in amino acid SEQ ID NOS: 1-12, 16, 18, 20, and 22.Preferably one of the chosen CDRs is from a V_(L) chain and the otherfrom a V_(H) chain.

In some aspects of this purification process the eluting solution has apH of about 3.3 to about 5.5. In other aspects, the pH of the elutingsolution is about 3.5 to about 5.5. Still other aspects comprise aneluting solution with a pH about 3.5 to about 4.2. Further aspects haveeluting solutions with a pH in the range of about 4.2 to about 5.5.

Another embodiment of the present invention comprises a method forevaluating physiological effects (e.g. anti-angiogenic properties)modulated by a humanized anti-α5β1 integrin antibody, which includesboth antibodies and Fab fragments. This method comprises providing aviable tissue sample capable of vascular regeneration; creating lesionsin the viable tissue sufficient to produce choroidal neovascularization;applying one or more doses of a humanized anti-α5β1 integrin antibody tothe viable tissue; and monitoring the dosed viable tissue forre-vascularization. In preferred embodiment, the method of evaluationincludes eye tissue as the viable tissue. In some embodiments the maculaof the eye is used. Also contemplated are methods of evaluating wherethe eye tissue used is that of a living primate (e.g. cynomologousmonkey).

In another embodiment, the method of evaluating comprises injecting achimeric or humanized anti-α5β1 integrin antibody intravitreally. In oneaspect of the invention, where two eyes of an individual are injured,injection of the antibodies in one eye results in antibodies contactinginjured tissue present in both eyes.

Another aspect of the method for evaluating physiological effectscomprises creating lesions by contacting the viable tissue with laserlight. This laser light can be from about 300 to about 700 mwatts, andthe exposure time is no more than 0.1 seconds, preferably less than 0.05seconds, and most preferably less than about 0.01 seconds. The lesionsshould be less than 200 μm, preferably less than 100 μm, more preferablyfrom about 50 to about 100 μm in diameter, and most preferably about 75to 25 μm in diameter.

Some aspects of the method include a monitoring step comprisingperiodically photographing the lesions treated by application of one ormore doses of a humanized anti-α5β1 integrin antibody. In other aspects,the monitoring step further comprises indirect ophthalmoscopicexamination of the posterior chamber of the eye, and biomicroscopicexamination of the anterior segment of the eye. In another aspect, themethod comprises a monitoring step that includes injecting intravenouslya fluorescein dye, and examining the viable tissue by fluoresceinangiography.

The method for evaluating physiological effects modulated by a chimericor humanized anti-α5β1 integrin antibody also includes an aspect whereinthe chimeric or humanized anti-α5β1 integrin antibody comprises avariable heavy chain region having a sequence 65%, preferably more than75%, more preferably 85%, 90%, 95%, 97% or 99% identical to an aminoacid sequence selected from the group consisting of SEQ ID NOS: 1-6, 16,20 and a variable light chain region independently selected and having asequence 65%, preferably more than 75%, more preferably 85%, 90%, 95%,97% or 99% identical to an amino acid sequence from the group consistingof SEQ ID NOS: 7-12, 18, 22. Most preferably, the humanized anti-α5β1integrin antibody comprises a variable heavy chain region having asequence selected from the group consisting of SEQ ID NOS: 1-6, 16, 20,and a variable light chain region independently selected from the groupconsisting of SEQ ID NOS: 7-12, 18, 22.

The method for evaluating physiological effects modulated by a chimericor humanized anti-α5β1 integrin antibody also includes an aspect whereinthe chimeric or humanized anti-α5β1 integrin antibody comprises avariable heavy chain region having a sequence selected from the groupconsisting of SEQ ID NOS: 2-6, 16, 20 and a variable light chain regionindependently selected from the group consisting of SEQ ID NOS: 8-12,18, 22.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 depicts the amino acid sequences (SEQ ID NOS: 1-12) for thevariable regions of the heavy (V_(H)) and light chains (V_(L)) of amurine anti-α5β1 integrin antibody (IIA1) and five humanized antibodiesderived from the murine original (1.0-5.0)

FIG. 2 depicts an alignment of the amino acid sequences from FIG. 1 (SEQID NOS: 1-12) that highlights sequence substitutions in the fivehumanized antibodies relative to the murine original (IIA1).

FIG. 3 depicts: (A) IIA1 V_(H) nucleic acid sequence (SEQ ID NO: 13) andamino acid sequence (SEQ ID NO: 46); (B) IIA1 V_(L) nucleic acidsequence (SEQ ID NO: 14) and amino acid sequence (SEQ ID NO: 47).

FIG. 4 depicts: (A) Antibody 200-4 V_(H) nucleic acid sequence (SEQ IDNO: 15) and amino acid sequence (SEQ ID NO: 16); (B) Antibody 200-4V_(L) nucleic acid sequence (SEQ ID NO: 17) and amino acid sequence (SEQID NO: 18).

FIG. 5 depicts: (A) M200 V_(H) nucleic acid sequence (SEQ ID NO: 19) andamino acid sequence (SEQ ID NO: 20); (B) M200 V_(L) nucleic acidsequence (SEQ ID NO: 21) and amino acid sequence (SEQ ID NO: 22).

FIG. 6 depicts the p200-M-H plasmid construct for expression of M200heavy chain.

FIG. 7 depicts the p200-M-L plasmid construct for expression of M200light chain.

FIG. 8 depicts the single plasmid p200-M for expression of M200 heavyand light chains.

FIG. 9 depicts the complete M200 heavy chain and light chain DNAsequences (SEQ ID NOS: 23-24).

FIG. 10 depicts the complete M200 heavy chain and light chain amino acidsequences (SEQ ID NOS: 25-26).

FIG. 11 depicts the complete F200 heavy chain DNA and amino acidsequences (SEQ ID NOS: 27-28).

FIG. 12 depicts the complete huM200 heavy chain and light chain DNAsequences (SEQ ID NOS: 29-30).

FIG. 13 depicts the complete huM200 heavy chain and light chain aminoacid sequences (SEQ ID NOS: 31-32).

FIG. 14 illustrates results of M200 is a potent inhibitor of endothelialcell growth, encompassing the anti-proliferative properties of ananti-VEGF mAb, HuMV833.

FIG. 15 illustrates results showing that M200 inhibits VEGF induced cellgrowth and inhibition of the M200 activity by anti-idiotype mAbs.

FIG. 16 illustrates results showing: (A), M200 induced cell deathvisualized by annexin staining; (B) quantification of annexin stainedcells by flow cytometry.

FIG. 17 illustrates results showing M200 causes increased cell death inproliferating versus senescent HUVEC.

FIG. 18 depicts results of in vitro tube formation assay for inhibitionof angiogenesis by F200.

FIG. 19 depicts fluorescein angiography images (FAs) of laser-inducedlesions in primate eyes at day 20 of treatment with (A) control(rituxan) and (B) M200.

FIG. 20 depicts fluorescein angiography images (FAs) of laser-inducedlesions in the left and right eyes of an individual primate at day 13 oftreatment with (A) control (left eye) and (B) M200 (right eye).

FIG. 21 depicts fluorescein angiography images (FAs) of laser-inducedlesions in the left and right eyes of an individual primate at day 20 oftreatment with (A) control (left eye) and (B) M200 (right eye).

FIG. 22 depicts fluorescein angiography images (FAs) of laser-inducedlesions in the left and right eyes of an individual primate at day 27 oftreatment with (A) control (left eye) and (B) M200 (right eye).

FIG. 23 depicts fluorescein angiography images (FAs) of laser-inducedlesions in the left and right eyes of an individual primate at day 13 oftreatment with (A) control (left eye) and (B) F200 (right eye).

FIG. 24 depicts fluorescein angiography images (FAs) of laser-inducedlesions in the left and right eyes of an individual primate at day 20 oftreatment with (A) control (left eye) and (B) F200 (right eye).

FIG. 25 depicts fluorescein angiography images (FAs) of laser-inducedlesions in the left and right eyes of an individual primate at day 27 oftreatment with (A) control (left eye) and (B) F200 (right eye).

FIG. 26 depicts results of a competition ELISA binding assay comparingbinding affinities of mouse antibody IIA1, chimeric antibody M200 (200-4EOS), and two humanized versions of M200: huM200-G4 and huM200-g2m3G.

FIG. 27 depicts representative fundus photographs and accompanying FAs(depicted as two smaller insets besides the larger fundus photo) forrabbit eyes from four groups of rabbits treated with M200 or F200following VEGF/bFGF Hydron™ Implant pellet (hydrogel polymer) asdescribed in Example 8. The clinical grading score (0-4 scale) isincluded on each fundus photograph.

FIG. 28 depicts four plots of results from the clinical grading andanalysis of fundus photographs (top two plots) and FAs (bottom twoplots) from the rabbit eye occult AMD model described in Example 8. Thesolid squares with dark solid line represent data for VEGF/bFGF withouttreatment; the upright solid triangles represent data for intravitrealM200 treatment; the inverted solid triangles represent data forintravitreal F200 treatment; the diamonds represent data for I.V. M200treatment; and the dashed line with solid circles represent data for atreatment of rabbits with a surgically implanted “blank” pellet.

DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the meaning commonly understood by a person skilled in the art towhich this invention belongs. The following references provide one ofskill with a general definition of many of the terms used in thisinvention: Singleton et al., Dictionary of Microbiology and MolecularBiology (2nd ed. 1994); The Cambridge Dictionary of Science andTechnology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R.Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, TheHarper Collins Dictionary of Biology (1991). As used herein, thefollowing terms have the meanings ascribed to them unless specifiedotherwise.

As used herein, “antibody” includes reference to an immunoglobulinmolecule immunologically reactive with a particular antigen, andincludes both polyclonal and monoclonal antibodies. The term alsoincludes genetically engineered forms such as chimeric antibodies (e.g.,humanized murine antibodies) and heteroconjugate antibodies (e.g.,bispecific antibodies). The term “antibody” also includes antigenbinding forms of antibodies, including fragments with antigen-bindingcapability (e.g., Fab′, F(ab′)₂, Fab, Fv and rIgG). See also, PierceCatalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.).See also, e.g., Kuby, J., Immunology, 3^(rd) Ed., W.H. Freeman & Co.,New York (1998). The term also refers to recombinant single chain Fvfragments (scFv). The term antibody also includes bivalent or bispecificmolecules, diabodies, triabodies, and tetrabodies. Bivalent andbispecific molecules are described in, e.g., Kostelny et al., (1992) JImmunol 148:1547, Pack and Pluckthun (1992) Biochemistry 31:1579,Hollinger et al., 1993, supra, Gruber et al. (1994) J Immunol :5368, Zhuet al. (1997) Protein Sci 6:781, Hu et al. (1996) Cancer Res. 56:3055,Adams et al. (1993) Cancer Res. 53:4026, and McCartney, et al. (1995)Protein Eng. 8:301.

An antibody immunologically reactive with a particular antigen can begenerated by recombinant methods such as selection of libraries ofrecombinant antibodies in phage or similar vectors, see, e.g., Huse etal., Science 246:1275-1281 (1989); Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotech. 14:309-314 (1996), or byimmunizing an animal with the antigen or with DNA encoding the antigen.

Typically, an immunoglobulin has a heavy and light chain. Each heavy andlight chain contains a constant region and a variable region, (theregions are also known as “domains”). Light and heavy chain variableregions contain four “framework” regions interrupted by threehypervariable regions, also called “complementarity-determining regions”or “CDRs”. The extent of the framework regions and CDRs have beendefined. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found.

References to “V_(H)” refer to the variable region of an immunoglobulinheavy chain of an antibody, including the heavy chain of an Fv, scFv, orFab. References to “V_(L)” refer to the variable region of animmunoglobulin light chain, including the light chain of an Fv, scFv,dsFv or Fab.

The phrase “single chain Fv” or “scFv” refers to an antibody in whichthe variable domains of the heavy chain and of the light chain of atraditional two chain antibody have been joined to form one chain.Typically, a linker peptide is inserted between the two chains to allowfor proper folding and creation of an active binding site.

A “chimeric antibody” is an immunoglobulin molecule in which (a) theconstant region, or a portion thereof, is altered, replaced or exchangedso that the antigen binding site (variable region) is linked to aconstant region of a different or altered class, effector functionand/or species, or an entirely different molecule which confers newproperties to the chimeric antibody, e.g., an enzyme, toxin, hormone,growth factor, drug, etc.; or (b) the variable region, or a portionthereof, is altered, replaced or exchanged with a variable region havinga different or altered antigen specificity.

A “humanized antibody” is an immunoglobulin molecule that containsminimal sequence derived from non-human immunoglobulin. Humanizedantibodies include human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a non-human species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. In some instances, Fv framework residues of thehuman immunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, a humanized antibody will comprise substantiallyall of at least one, and typically two, variable domains, in which allor substantially all of the CDR regions correspond to those of anon-human immunoglobulin and all or substantially all of the framework(FR) regions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin (Jones et al., Nature 321:522-525 (1986); Riechmann etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992)). Humanization can be essentially performed followingthe method of Winter and co-workers (Jones et al., Nature 321:522-525(1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al.,Science 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody.Accordingly, such humanized antibodies are chimeric antibodies (U.S.Pat. No. 4,816,567), wherein substantially less than an intact humanvariable domain has been substituted by the corresponding sequence froma non-human species.

“Epitope” or “antigenic determinant” refers to a site on an antigen towhich an antibody binds. Epitopes can be formed both from contiguousamino acids or noncontiguous amino acids juxtaposed by tertiary foldingof a protein. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed (1996).

“pH-sensitive anti-α5β1 integrin antibody” refers to antibodies thatspecifically recognize α5β1 integrin, and precipitate from solution whensubjected to immunopurification using α5β1 integrin as the ligand atneutral or basic pH. pH-sensitive anti-α5β1 integrin antibodiestypically comprise two or more CDR sequences chosen independently fromany of the V_(H) or V_(L) sequences depicted in FIG. 1.

“Angiogenesis” and “neoangiogenesis” refer to the formation of new bloodvessels, typically in response to insult, injury or disease. For thepurposes of this application, the term “injury,” and grammaticalvariations of the same, includes insult, disease, or other event thatresults in a tissue response which includes angiogenesis. Angiogenesisalso occurs in tumor formation and metastasis, and during embryogenesis,growth and development of higher animals.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, preferably 70%, 75%, 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specifiedregion, when compared and aligned for maximum correspondence over acomparison window or designated region) as measured using a BLAST orBLAST 2.0 sequence comparison algorithms with default parametersdescribed below, or by manual alignment and visual inspection (see,e.g., NCBI web site www.ncbi.nlm.nih.gov/BLAST/ or the like). Suchsequences are then said to be “substantially identical.” This definitionalso refers to, or may be applied to, the complement of a test sequence.The definition also includes sequences that have deletions and/oradditions, as well as those that have substitutions, as well asnaturally occurring, e.g., polymorphic or allelic variants, and man-madevariants. As described below, the preferred algorithms can account forgaps and the like. Preferably, identity exists over a region that is atleast about 25 amino acids or nucleotides in length, or more preferablyover a region that is 50-100 amino acids or nucleotides in length.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Preferably,default program parameters can be used, or alternative parameters can bedesignated. The sequence comparison algorithm then calculates thepercent sequence identities for the test sequences relative to thereference sequence, based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof one of the number of contiguous positions selected from the groupconsisting typically of from 20 to 600, usually about 50 to about 200,more usually about 100 to about 150 in which a sequence may be comparedto a reference sequence of the same number of contiguous positions afterthe two sequences are optimally aligned. Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group, 575 Science Dr.,Madison, Wis.), or by manual alignment and visual inspection (see, e.g.,Current Protocols in Molecular Biology (Ausubel et al., eds. 1995supplement)).

Preferred examples of algorithms that are suitable for determiningpercent sequence identity and sequence similarity include the BLAST andBLAST 2.0 algorithms, which are described in Altschul et al., Nucl.Acids Res. 25:3389-3402 (1977) and Altschul et al., J. Mol. Biol.215:403-410 (1990). BLAST and BLAST 2.0 are used, with the parametersdescribed herein, to determine percent sequence identity for the nucleicacids and proteins of the invention. Software for performing BLASTanalyses is publicly available through the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov). This algorithminvolves first identifying high scoring sequence pairs (HSPs) byidentifying short words of length W in the query sequence, which eithermatch or satisfy some positive-valued threshold score T when alignedwith a word of the same length in a database sequence. T is referred toas the neighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, e.g.,for nucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always>0) and N (penalty score for mismatchingresidues; always<0). For amino acid sequences, a scoring matrix is usedto calculate the cumulative score. Extension of the word hits in eachdirection are halted when: the cumulative alignment score falls off bythe quantity X from its maximum achieved value; the cumulative scoregoes to zero or below, due to the accumulation of one or morenegative-scoring residue alignments; or the end of either sequence isreached. The BLAST algorithm parameters W, T, and X determine thesensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915 (1989))alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001. Log valuesmay be large negative numbers, e.g., 5, 10, 20, 30, 40, 40, 70, 90, 110,150, 170, etc.

An indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the antibodiesraised against the polypeptide encoded by the second nucleic acid, asdescribed below. Thus, a polypeptide is typically substantiallyidentical to a second polypeptide, e.g., where the two peptides differonly by conservative substitutions. Another indication that two nucleicacid sequences are substantially identical is that the two molecules ortheir complements hybridize to each other under stringent conditions, asdescribed below. Yet another indication that two nucleic acid sequencesare substantially identical is that the same primers can be used toamplify the sequences.

The terms “isolated,” “purified,” or “biologically pure” refer tomaterial that is substantially or essentially free from components thatnormally accompany it as found in its native state. Purity andhomogeneity are typically determined using analytical chemistrytechniques such as polyacrylamide gel electrophoresis or highperformance liquid chromatography. A protein or nucleic acid that is thepredominant species present in a preparation is substantially purified.In particular, an isolated nucleic acid is separated from some openreading frames that naturally flank the gene and encode proteins otherthan protein encoded by the gene. The term “purified” in someembodiments denotes that a nucleic acid or protein gives rise toessentially one band in an electrophoretic gel. Preferably, it meansthat the nucleic acid or protein is at least 85% pure, more preferablyat least 95% pure, and most preferably at least 99% pure. “Purify” or“purification” in other embodiments means removing at least onecontaminant from the composition to be purified. In this sense,purification does not require that the purified compound be homogenous,e.g., 100% pure.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers, those containing modified residues, and non-naturallyoccurring amino acid polymer.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction similarly to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, as well asthose amino acids that are later modified, e.g., hydroxyproline,γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers tocompounds that have the same basic chemical structure as a naturallyoccurring amino acid, e.g., an α carbon that is bound to a hydrogen, acarboxyl group, an amino group, and an R group, e.g., homoserine,norleucine, methionine sulfoxide, methionine methyl sulfonium. Suchanalogs may have modified R groups (e.g., norleucine) or modifiedpeptide backbones, but retain the same basic chemical structure as anaturally occurring amino acid. Amino acid mimetics refers to chemicalcompounds that have a structure that is different from the generalchemical structure of an amino acid, but that functions similarly to anaturally occurring amino acid.

Amino acids may be referred to herein by their commonly known threeletter symbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical or associated, e.g., naturallycontiguous, sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode mostproteins. For instance, the codons GCA, GCC, GCG and GCU all encode theamino acid alanine. Thus, at every position where an alanine isspecified by a codon, the codon can be altered to another of thecorresponding codons described without altering the encoded polypeptide.Such nucleic acid variations are “silent variations,” which are onespecies of conservatively modified variations. Every nucleic acidsequence herein which encodes a polypeptide also describes silentvariations of the nucleic acid. One of skill will recognize that incertain contexts each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, often silent variations of a nucleicacid which encodes a polypeptide is implicit in a described sequencewith respect to the expression product, but not with respect to actualprobe sequences.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention. Typically conservativesubstitutions for one another include e.g.: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

A “label” or a “detectable moiety” is a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, chemical, orother physical means. For example, useful labels include fluorescentdyes, electron-dense reagents, enzymes (e.g., as commonly used in anELISA), biotin, digoxigenin, or haptens and proteins or other entitieswhich can be made detectable, e.g., by incorporating a radiolabel intothe peptide or used to detect antibodies specifically reactive with thepeptide. The radioisotope may be, for example, 3H, 14C, 32P, 35S, or125I.

In some cases, particularly using anti-α5β1 integrin antibodies, theradioisotopes are used as toxic moieties, as described below. The labelsmay be incorporated into the antibodies at any position. Any methodknown in the art for conjugating the antibody to the label may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982). The lifetime of radiolabeled peptides orradiolabeled antibody compositions may extended by the addition ofsubstances that stabilize the radiolabeled peptide or antibody andprotect it from degradation. Any substance or combination of substancesthat stabilize the radiolabeled peptide or antibody may be usedincluding those substances disclosed in U.S. Pat. No. 5,961,955.

“Antibody affinity matrix” refers to any material capable ofpreferentially binding an antibody. Antibody affinity matrix materialsinclude polypeptides, polysaccharides, fatty acids, lipids, nucleicacids, including aptamers, or conjugates of these (e.g., glycoproteins,lipoproteins, glycolipids). In certain instances antibody affinitymatrix materials can be a macromolecular structure such as amultiprotein complex, a biological membrane or a virus. Other examplesof antibody affinity matrix materials are protein A, protein G, lectins,and Fc receptors.

“Protein A” refers to a highly stable surface receptor produced byStaphylococcus aureus, which is capable of binding the Fc portion ofimmunoglobulins, especially IgGs, from a large number of species (Boyle,M. D. P. and K. J. Reis. Bacterial Fc Receptors. Biotechnology 5:697-703(1987).). One protein A molecule can bind at least 2 molecules of IgGsimultaneously (Sjoquist, J., Meloun, B. and Hjelm, H. Protein Aisolated from Staphylococcus aureus after digestion with lysostaphin.Eur J Biochem 29: 572-578 (1972)).

“Protein G” refers to a cell surface-associated protein fromstreptococcus that binds to IgG with high affinity. It has three highlyhomologous IgG-binding domains. (See Lian, et al. 1992. Journal of Mol.Biol. 228:1219-1234 and Derrick and Wigley. 1994. Journal of Mol. Biol.243:906-918.)

The term “recombinant” when used with reference, e.g., to a cell, ornucleic acid, protein, or vector, indicates that the cell, nucleic acid,protein or vector, has been modified by the introduction of aheterologous nucleic acid or protein or the alteration of a nativenucleic acid or protein, or that the cell is derived from a cell somodified. Thus, e.g., recombinant cells express genes that are not foundwithin the native (non-recombinant) form of the cell or express nativegenes that are otherwise abnormally expressed, under expressed or notexpressed at all. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid, e.g., using polymerases and endonucleases,in a form not normally found in nature. In this manner, operably linkageof different sequences is achieved. Thus an isolated nucleic acid, in alinear form, or an expression vector formed in vitro by ligating DNAmolecules that are not normally joined, are both considered recombinantfor the purposes of this invention. It is understood that once arecombinant nucleic acid is made and reintroduced into a host cell ororganism, it will replicate non-recombinantly, i.e., using the in vivocellular machinery of the host cell rather than in vitro manipulations;however, such nucleic acids, once produced recombinantly, althoughsubsequently replicated non-recombinantly, are still consideredrecombinant for the purposes of the invention. Similarly, a “recombinantprotein” is a protein made using recombinant techniques, i.e., throughthe expression of a recombinant nucleic acid as depicted above.

The term “heterologous” when used with reference to portions of anucleic acid indicates that the nucleic acid comprises two or moresubsequences that are not normally found in the same relationship toeach other in nature. For instance, the nucleic acid is typicallyrecombinantly produced, having two or more sequences, e.g., fromunrelated genes arranged to make a new functional nucleic acid, e.g., apromoter from one source and a coding region from another source.Similarly, a heterologous protein will often refer to two or moresubsequences that are not found in the same relationship to each otherin nature (e.g., a fusion protein).

A “promoter” is defined as an array of nucleic acid control sequencesthat direct transcription of a nucleic acid. As used herein, a promoterincludes necessary nucleic acid sequences near the start site oftranscription, such as, in the case of a polymerase II type promoter, aTATA element. A promoter also optionally includes distal enhancer orrepressor elements, which can be located as much as several thousandbase pairs from the start site of transcription. A “constitutive”promoter is a promoter that is active under most environmental anddevelopmental conditions. An “inducible” promoter is a promoter that isactive under environmental or developmental regulation. The term“operably linked” refers to a functional linkage between a nucleic acidexpression control sequence (such as a promoter, or array oftranscription factor binding sites) and a second nucleic acid sequence,wherein the expression control sequence directs transcription of thenucleic acid corresponding to the second sequence.

An “expression vector” is a nucleic acid construct, generatedrecombinantly or synthetically, with a series of specified nucleic acidelements that permit transcription of a particular nucleic acid in ahost cell. The expression vector can be part of a plasmid, virus, ornucleic acid fragment. Typically, the expression vector includes anucleic acid to be transcribed operably linked to a promoter.

The phrase “specifically (or selectively) binds” to an antibody or“specifically (or selectively) immunoreactive with,” when referring to aprotein or peptide, refers to a binding reaction that is determinativeof the presence of the protein, in a heterogeneous population ofproteins and other biologics. Thus, under designated immunoassayconditions, the specified antibodies bind to a particular proteinsequences at least two times the background and more typically more than10 to 100 times background.

Specific binding to an antibody under such conditions requires anantibody that is selected for its specificity for a particular protein.For example, antibodies raised against a particular protein, polymorphicvariants, alleles, orthologs, and conservatively modified variants, orsplice variants, or portions thereof, can be selected to obtain onlythose polyclonal antibodies that are specifically immunoreactive withα5β1 integrin and not with other proteins. This selection may beachieved by subtracting out antibodies that cross-react with othermolecules. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein. Forexample, solid-phase ELISA immunoassays are routinely used to selectantibodies specifically immunoreactive with a protein (see, e.g., Harlow& Lane, Antibodies, A Laboratory Manual (1988) for a description ofimmunoassay formats and conditions that can be used to determinespecific immunoreactivity).

“Cancer cells,” “transformed” cells or “transformation” in tissueculture, refers to spontaneous or induced phenotypic changes that do notnecessarily involve the uptake of new genetic material. Althoughtransformation can arise from infection with a transforming virus andincorporation of new genomic DNA, or uptake of exogenous DNA, it canalso arise spontaneously or following exposure to a carcinogen, therebymutating an endogenous gene. Transformation is associated withphenotypic changes, such as immortalization of cells, aberrant growthcontrol, nonmorphological changes, and/or malignancy (see, Freshney,Culture of Animal Cells a Manual of Basic Technique (3rd ed. 1994)).

II. Introduction

The present invention provides chimeric and humanized anti-α5β1 integrinantibodies with improved properties over existing anti-α5β1 integrinantibodies. The present invention also provides pharmaceuticalcompositions comprising the new antibodies, and improved methods fortreating disease states and injuries to tissues that are exacerbated byangiogenesis.

The chimeric and humanized antibodies of the invention have a longerhalf-life and are less antigenic when administered to a human being thanexisting forms. The improvement is illustrated diagrammatically in FIG.2, and involves altering framework and constant regions of murineanti-α5β1 integrin (IIA1) antibodies to “humanize” them.

Humanized antibodies generally have at least three potential advantagesfor use in human therapy. First, it may interact better with the humanimmune system, e.g., to destroy target cells more efficiently bycomplement-dependent cytotoxicity (CDC) or antibody-dependent cellularcytotoxicity (ADCC). Second, the human immune system should notrecognize the antibody as foreign. Third, the half-life in the humancirculation will be similar to naturally occurring human antibodies,allowing smaller and less frequent doses to be given.

Structurally, humanized antibodies generally have constant and framework(FR) regions that are of human origin, and complementary domain regions(CDRs) that originate from the antibody of the animal in which theanti-α5β1 integrin antibody was raised.

Structurally, chimeric antibodies generally have variable-chain regionsoriginating from the antibody of the animal in which the anti-α5β1integrin antibody was raised, and constant chain regions of humanorigin.

Functionally both chimeric and humanized anti-α5β1 integrin antibodiesspecifically recognize α5β1 integrin, and prevent α5β1 integrin frominteracting with its receptor.

Various methods for preparing humanized and chimeric anti-α5β1 integrinantibodies are provided herein. “Humanized” antibodies are generallychimeric or mutant monoclonal antibodies from mouse, rat, hamster,rabbit or other species, bearing human constant and/or variable regiondomains or specific changes. Techniques for generating “humanized” and“chimeric” anti-α5β1 integrin antibodies are well known to those ofskill in the art and may be found in literature reference and patentscited herein.

III. Preparation of Recombinant Chimeric and Humanized Anti-α5β1Integrin Antibodies, and Fab Fragments Derived Therefrom

Antibodies of the present invention are prepared by immunizing an animalwith α5β1 integrin, or a peptide derived therefrom to induce anti-α5β1integrin antibody production. Lymphoid tissue expressing the antibodiesis then isolated and the nucleic acids encoding the heavy and lightchains of the anti-α5β1 integrin antibodies are purified. The purifiednucleic acids are then recombinantly manipulated (according to methodswell-known in the art) to create nucleic acids encoding chimeric,humanized, single-chain, Fab or Fab₂ antibodies that specificallyrecognize α5β1 integrin.

The recombinantly manipulated nucleic acids are then used to createanti-α5β1 integrin antibody-producing cells. These cells producemonoclonal antibodies that inhibit, or prevent, α5β1 integrin frombinding to its receptor, which results in inhibition of angiogenesis insusceptible tissue.

A. Production of Cells Producing, and Nucleic Acids Encoding, Anti-α5β1Integril Antibodies

In order to prepare recombinant chimeric and humanized anti-α5β1integrin antibodies, the nucleic acid encoding non-human anti-α5β1integrin antibodies must first be isolated. This is typically done byimmunizing an animal, for example a mouse, with prepared α5β1 integrinor an antigenic peptide derived therefrom. Typically mice are immunizedtwice intraperitoneally with approximately 50 micrograms of proteinantibody per mouse. Sera from immunized mice can be tested for antibodyactivity by immunohistology or immunocytology on any host systemexpressing such polypeptide and by ELISA with the expressed polypeptide.For immunohistology, active antibodies of the present invention can beidentified using a biotin-conjugated anti-mouse immunoglobulin followedby avidin-peroxidase and a chromogenic peroxidase substrate.Preparations of such reagents are commercially available; for example,from Zymed Corp., San Francisco, Calif. Mice whose sera containdetectable active antibodies according to the invention can besacrificed three days later and their spleens removed for fusion andhybridoma production. Positive supernatants of such. hybridomas can beidentified using the assays common to those of skill in the art, forexample, Western blot analysis.

The nucleic acids encoding the desired antibody chains can then beisolated by, for example, using hybridoma mRNA or splenic mRNA as atemplate for PCR amplification of the heavy and light chain genes [Huse,et al., Science 246:1276 (1989)]. Nucleic acids for producing bothantibodies and intrabodies can be derived from murine monoclonalhybridomas using this technique [Richardson J. H., et al., Proc. Natl.Acad. Sci. USA 92:3137-3141 (1995); Biocca S., et al., Biochem andBiophys Res Comm, 197:422-427 (1993) Mhashilkar, A. M., et al., EMBO J14:1542-1551 (1995)]. These hybridomas provide a reliable source ofwell-characterized reagents for the construction of antibodies and areparticularly useful once their epitope reactivity and affinity has beencharacterized. Isolation of nucleic acids from isolated cells isdiscussed further in Clackson, T., et al., Nature 352:624-628 (1991)(spleen) and Portolano, S., et al., supra; Barbas, C. F., et al., supra;Marks, J. D., et al., supra; Barbas, C. F., et al., Proc Natl Acad SciUSA 88:.7978-7982 (1991) (human peripheral blood lymphocytes).

B. Creating Recombinant Antibodies

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues form a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods of generating humanized antibodies are well-known in the art andfully described elsewhere. See, e.g., Queen et al., U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (each of which isincorporated by reference in its entirety). Antibodies can be humanizedusing a variety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Mol. Immunol., 28:489-498 (1991); Studnickaet al., Prot. Eng. 7:805-814 (1994); Roguska et al., Proc. Natl. Acad.Sci. 91:969-973 (1994), and chain shuffling (U.S. Pat. No. 5,565,332),all of which are hereby incorporated by reference in their entireties.

A number of methods have been described to produce recombinant chimericantibodies. Controlled rearrangement of antibody domains joined throughprotein disulfide bonds to form chimeric antibodies can be utilized(Konieczny et al., Haematologia, 14(1):95-99, 1981). Recombinant DNAtechnology can also be used to construct gene fusions between DNAsequences encoding mouse antibody variable light and heavy chain domainsand human antibody light and heavy chain constant domains. See e.g.,Morrison et al., Proc. Natl. Acad. Sci. USA, 81(21): 6851-6855, 1984;Morrison, Science 229:1202-1207 (1985); Oi et al., BioTechniques4:214-221 (1986); Gillies et al., J. Immunol. Methods 125:191-202(1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which areincorporated herein by reference in their entireties.

DNA sequences encoding the antigen binding portions or complementaritydetermining regions (CDRs) of murine monoclonal antibodies can begrafted by molecular means into the DNA sequences encoding theframeworks of human antibody heavy and light chains (Jones et al.,Nature, 321(6069):522-525, 1986.; Riechmann et al., Nature,332(6162):323-327, 1988.). The expressed recombinant products are called“reshaped” or humanized antibodies, and comprise the framework of ahuman antibody light or heavy chain and the antigen recognitionportions, CDRs, of a murine monoclonal antibody.

Another method for producing humanized antibodies is described in U.S.Pat. No. 5,639,641, incorporated herein by reference. The methodprovides, via resurfacing, humanized rodent antibodies that haveimproved therapeutic efficacy due to the presentation of a human surfacein the variable region. In the method: (1) position alignments of a poolof antibody heavy and light chain variable regions is generated to givea set of heavy and light chain variable region framework surface exposedpositions, wherein the alignment positions for all variable regions areat least about 98% identical; (2) a set of heavy and light chainvariable region framework surface exposed amino acid residues is definedfor a rodent antibody (or fragment thereof); (3) a set of heavy andlight chain variable region framework surface exposed amino acidresidues that is most closely identical to the set of rodent surfaceexposed amino acid residues is identified; (4) the set of heavy andlight chain variable region framework surface exposed amino acidresidues defined in step (2) is substituted with the set of heavy andlight chain variable region framework surface exposed amino acidresidues identified in step (3), except for those amino acid residuesthat are within 5 Å of any atom of any residue of the complementaritydetermining regions of the rodent antibody; and (5) the humanized rodentantibody having binding specificity is produced.

A similar method for the production of humanized antibodies is describedin U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and 5,530,101, eachincorporated herein by reference. These methods involve producinghumanized immunoglobulins having one or more complementarity determiningregions (CDRs) and possible additional amino acids from a donorimmunoglobulin and a framework region from an accepting humanimmunoglobulin. Each humanized immunoglobulin chain usually comprises,in addition to the CDRs, amino acids from the donor immunoglobulinframework that are capable of interacting with the CDRs to effectbinding affinity, such as one or more amino acids that are immediatelyadjacent to a CDR in the donor immunoglobulin or those within about 3 Å,as predicted by molecular modeling. The heavy and light chains may eachbe designed by using any one, any combination, or all of the variousposition criteria described in U.S. Pat. Nos. 5,693,762; 5,693,761;5,585,089; and 5,530,101. When combined into an intact antibody, thehumanized immunoglobulins are substantially non-antibodyic in humans andretain substantially the same affinity as the donor immunoglobulin tothe original antigen.

An additional method for producing humanized antibodies is described inU.S. Pat. Nos. 5,565,332 and 5,733,743, each incorporated herein byreference. This method combines the concept of humanizing antibodieswith the phagemid libraries also described in detail herein. In ageneral sense, the method utilizes sequences from the antigen bindingsite of an antibody or population of antibodies directed against anantigen of interest. Thus for a single rodent antibody, sequencescomprising part of the antigen binding site of the antibody may becombined with diverse repertoires of sequences of human antibodies thatcan, in combination, create a complete antigen binding site.

The antigen binding sites created by this process differ from thosecreated by CDR grafting, in that only the portion of sequence of theoriginal rodent antibody is likely to make contacts with antigen in asimilar manner. The selected human sequences are likely to differ insequence and make alternative contacts with the antigen from those ofthe original binding site. However, the constraints imposed by bindingof the portion of original sequence to antigen and the shapes of theantigen and its antigen binding sites, are likely to drive the newcontacts of the human sequences to the same region or epitope of theantigen. This process has therefore been termed “epitope imprintedselection” (EIS).

Starting with an animal antibody, one process results in the selectionof antibodies that are partly human antibodies. Such antibodies may besufficiently similar in sequence to human antibodies to be used directlyin therapy or after alteration of a few key residues. Sequencedifferences between the rodent component of the selected antibody withhuman sequences could be minimized by replacing those residues thatdiffer with the residues of human sequences, for example, by sitedirected mutagenesis of individual residues, or by CDR grafting ofentire loops. However, antibodies with entirely human sequences can alsobe created. EIS therefore offers a method for making partly human orentirely human antibodies that bind to the same epitope as animal orpartly human antibodies respectively. In EIS, repertoires of antibodyfragments can be displayed on the surface of filamentous phase and thegenes encoding fragments with antigen binding activities selected bybinding of the phage to antigen.

Additional methods for humanizing antibodies contemplated for use in thepresent invention are described in U.S. Pat. Nos. 5,750,078; 5,502,167;5,705,154; 5,770,403; 5,698,417; 5,693,493; 5,558,864; 4,935,496; and4,816,567, each incorporated herein by reference.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain humanizedantibodies to α5β1 integrin.

C. Expressing Recombinant Chimeric or Humanized Antibodies

The resultant antibody can be expressed through one or more vectorscomprising nucleic acids encoding the antibody.

Preferably the nucleic acid segments encoding the heavy and light chainsof the antibody are in a single transcriptional unit, with translationof one of the coding nucleic acids under the control of an IRESsequence. Vectors include chemical conjugates such as described in WO93/64701, which has targeting moiety (e.g. a ligand to a cellularsurface receptor), and a nucleic acid binding moiety (e.g. polylysine),viral vector (e.g. a DNA or RNA viral vector), fusion proteins such asdescribed in PCT/US 95/02140 (WO 95/22618) which is a fusion proteincontaining a target moiety (e.g. an antibody specific for a target cell)and a nucleic acid binding moiety (e.g. a protamine), plasmids, phage,etc. The vectors can be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector [Geller, A. I. et al., J. Neurochem, 64:487(1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover,Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al.,Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al.,Proc Natl. Acad. Sci USA 87:1149 (1990)], Adenovirus Vectors [LeGalLaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995)] andAdeno-associated Virus Vectors [Kaplitt, M. G. et al., Nat. Genet. 8:148(1994)].

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofecton, cell microinjection, andviral vectors.

The vector can be employed to target essentially any desired targetcell, such as a glioma. For example, stereotaxic injection can be usedto direct the vectors (e.g. adenovirus, HSV) to a desired location.Additionally, the particles can be delivered by intracerebroventricular(icv) infusion using a minipump infusion system, such as a SynchroMedInfusion System. A method based on bulk flow, termed convection, hasalso proven effective at delivering large molecules to extended areas ofthe brain and may be useful in delivering the vector to the target cell(Bobo et al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrisonet al., Am. J. Physiol. 266:292-305 (1994)). Other methods that can beused include catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

D. Isolation and Characterization of Recombinant Antibodies

As used herein, the term “antibodies” includes polyclonal antibodies,affinity-purified polyclonal antibodies, monoclonal antibodies, andantigen-binding fragments, such as F(ab′)₂ and Fab proteolyticfragments. Genetically engineered intact antibodies or fragments, suchas chimeric antibodies, Fv fragments, single chain antibodies and thelike, as well as synthetic antigen-binding peptides and polypeptides,are also included. Non-human antibodies may be humanized by graftingonly non-human CDRs onto human framework and constant regions, or byincorporating the entire non-human variable domains (optionally“cloaking” them with a human-like surface by replacement of exposedresidues, wherein the result is a “veneered” antibody). In someinstances, humanized antibodies may retain non-human residues within thehuman variable region framework domains to enhance proper bindingcharacteristics. Through humanizing antibodies, biological half-life maybe increased, and the potential for adverse immune reactions uponadministration to humans is reduced. Alternative techniques forgenerating or selecting antibodies useful herein include in vitroexposure of lymphocytes to α5β1 integrin protein or peptide, andselection of antibody display libraries in phage or similar vectors (forinstance, through use of immobilized or labeled α5β1 integrin protein orpeptide).

E. Affinity Purification

Affinity purification of an antibody pool or sera provides apractitioner with a more uniform reagent. Methods for enrichinganti-α5β1 integrin antibodies using antibody affinity matrices to forman affinity column are well known in the art and available commercially(AntibodyShop, c/o Statens Serum Institut, Artillerivej 5, Bldg. P2,DK-2300 Copenhagen S). Briefly, an antibody affinity matrix is attachedto an affinity support (see e.g.; CNBR SEPHAROSE® (gel filtrationmatrix), Pharmacia Biotech). A mixture comprising antibodies is thenpassed over the affinity matrix, to which the antibodies bind. Boundantibodies are released by techniques common to those familiar with theart, yielding a concentrated antibody pool. The enriched antibody poolcan then be used for further immunological studies, some of which aredescribed herein by way of example. Although the antibody affinitymatrices used to isolate the antibodies of the present invention are notdesigned to specifically recognize the anti-α5β1 integrin antibodies ofthe present invention, this does not limit the utility of the affinitymatrices in purifying the antibodies, as the antibodies are expressed asrecombinant proteins in systems that are monoclonal in their nature.

Isolated anti-α5β1 integrin antibodies can be used in a competitivebinding immunoassay as described above to compare a second protein,thought to be perhaps a variant of α5β1 integrin. In order to make thiscomparison, the two proteins are each assayed at a wide range ofconcentrations and the amount of each protein required to inhibit 50% ofthe binding of the antisera to the immobilized protein is determined. Ifthe amount of the second protein required to inhibit 50% of binding isless than 10 times the amount of α5β1 integrin required to inhibit 50%of binding, then the second protein is said to specifically bind to theantibodies generated to the α5β1 integrin.

F. pH-Sensitive Antibody Purification.

Some antibodies of the present invention displayed a propensity toprecipitate when affinity purified at neutral or basic pH. To addressthis issue, another aspect of the invention relates to a process forpurification of pH-sensitive antibodies, including the antibodiescomprising to the amino acid sequences indicated in FIGS. 1-5, 10, 11and 13 and chimeric antibodies that include the mouse variable region orhave 80% or more sequence identity with the mouse variable region, orhaving 80% or more sequence identity to the CDR regions of theantibodies included in FIGS. 1-5. The process comprises generallyconducting an affinity chromatography for the antibody using achromatographic column, e.g. an ion exchange column, that contains boundAntibody affinity matrix, followed by elution of the antibody at a pH offrom about 3.0 to about 5.5, preferably from about 3.3 to about 5.5, andmost preferably either from about 3.5 to about 4.2 or from about 4.2 toabout 5.5. Lower pH values within this range are more suitable forsmall-scale purification while a pH of about 4.2 or higher is consideredmore suitable for larger scale operations. Operation of the purificationprocess within this range produces a product with little or noaggregation, most preferably with essentially no aggregation.

Affinity chromatography is one means known in the art for isolating orpurifying a substance, such as an antibody or other biologically activemacromolecule. This is accomplished in general by passing a solutioncontaining the antibody through a chromatographic column that containsone or more ligands that specifically bind to the antibody immobilizedon the column. Such groups can extract the antibody from the solutionthrough ligand-affinity reactions. Once that is accomplished, theantibody may be recovered by elution from the column.

This aspect of the invention therefore comprises a method for thepurification of anti-α5β1 integrin antibodies using an antibody affinitymatrix bound to a substrate, wherein the improvement comprises elutingthe antibodies from the substrate-bound antibody affinity matrix usingan eluting solution having a pH of from about 3.0 to about 5.5.

More specifically, this aspect of the invention comprises a method forthe purification of anti-α5β1 integrin antibodies comprising: (a)absorbing the antibody onto antibody affinity matrix bound to asubstrate; and (b) eluting the antibody from the substrate-boundantibody affinity matrix using an eluting solution having a pH of fromabout 3.0 to about 5.5. In some embodiments, the process also includesthe step of (c) recovering the purified antibody.

However, when antibody is to be further purified or treated, then aspecific recovery step may not be necessary at this point.

The purification process involves the absorption of the antibodies ontoantibody affinity matrix bound to a substrate. Various forms of antibodyaffinity matrix may be used. The only requirement is that the antibodyaffinity matrix molecule possesses the ability to bind the antibody thatis to be purified. For example, antibody affinity matrix isolated fromnatural sources, antibody affinity matrix produced by recombinant DNAtechniques, modified forms of antibody affinity matrices, or fragmentsof these materials which retain binding ability for the antibody inquestion may be employed. Exemplary materials for use as antibodyaffinity matrices include polypeptides, polysaccharides, fatty acids,lipids, nucleic acid aptamers, glycoproteins, lipoproteins, glycolipids,multiprotein complexes, a biological membrane, viruses, protein A,protein G, lectins, and Fc receptors.

The antibody affinity matrix is attached to a solid phase or support bya general interaction (for example, by non-specific, ion exchangebonding, by hydrophobic/hydrophilic interactions), or by a specificinteraction (for example, antigen-antibody interaction), or by covalentbonding between the ligand and the solid phase. Alternately, anintermediate compound or spacer can be attached to the solid phase andthe antibody affinity matrix can then be immobilized on the solid phaseby attaching the affinity matrix to the spacer. The spacer can itself bea ligand (i.e., a second ligand) that has a specific binding affinityfor the free antibody affinity matrix.

The antibody affinity matrix can be attached to various substrates orsupports. Typically, ion exchange or coupling (e.g., CNBr-activated)resins are used for this purpose. The antibodies may be adsorbed ontothe substrate-bound antibody affinity matrix using various procedures.Preferably, a column procedure is employed, and the antibodies areadsorbed to the column using a buffer solution prepared with anappropriate buffer. Typical buffers and operating conditions are wellknown in the art.

The antibodies may be eluted from the substrate-bound antibody affinitymatrix using conventional procedures, e.g. eluting the antibodies fromthe column using a buffer solution. To minimize precipitation,pH-sensitive anti-α5β1 integrin antibodies are preferably eluted with abuffer solution comprising 0.1 M glycine at pH 3.5. To minimizedegradation and/or denaturation, the temperature of the buffer solutionis preferably kept below 10° C., more preferably at or below 4° C. Forthe same reasons, the period during which the antibodies are exposed toacidic pH should also be minimized. This is accomplished, for example,by adding a predetermined amount of a basic solution to the elutedantibody solution. Preferably this basic solution is a bufferedsolution, more preferably a volatile basic buffered solution, mostpreferably an ammonia solution.

The elution of antibodies from the substrate-bound antibody affinitymatrix may be monitored by various methods well-known in the art. Forexample, if column procedures are employed, fractions may be collectedfrom the columns, and the presence of protein determined by measuringthe absorption of the fractions. If antibodies of known specificity arebeing purified, the presence of the antibodies in fractions collectedfrom the columns may be measured by immunoassay techniques, for example,radioimmunoassay (RIA) or enzyme immunoassay (EIA).

The process of the present invention may be performed at any convenienttemperature which does not substantially degrade the antibody beingpurified, or detrimentally affect the antibody affinity matrix bound toa substrate. Preferably, the temperature employed is room temperature.The antibodies eluted from the antibody affinity matrix column may berecovered, if desired, using various methods known in the art.

G. Avidity Testing

Avidity testing allows one skilled in the art to identify antibodiesspecifically recognizing one or more epitopes of α5β1 integrin.Antibodies are defined to be specifically binding if: 1) they exhibit athreshold level of binding activity, and/or 2) they do not significantlycross-react with related polypeptide molecules. First, antibodies hereinspecifically bind if they bind to a α5β1 integrin polypeptide, peptideor epitope with a binding affinity (K_(a)) of 10⁶ mol⁻¹ or greater,preferably 10⁷ mol⁻¹ or greater, more preferably 10⁸ mol⁻¹ or greater,and most preferably 10⁹ mol⁻¹ or greater. The binding affinity of anantibody can be readily determined by one of ordinary skill in the art,for example, by Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660-72, 1949), or by surface plasmon resonance using BIAcore.

Second, antibodies specifically bind if they do not significantlycross-react with related polypeptides. Antibodies do not significantlycross-react with related polypeptide molecules, for example, if theydetect α5β1 integrin polypeptide but not known related polypeptidesusing a standard Western blot analysis (Ausubel et al., ibid.). Examplesof known related polypeptides are orthologs, proteins from the samespecies that are members of the integrin family of proteins, thepolypeptides shown in alignment FIG. 1, mutant α5β1 integrinpolypeptides, and the like. Moreover, antibodies may be “screenedagainst” known related polypeptides to isolate a population thatspecifically binds to the α5β1 integrin. For example, antibodies raisedto human α5β1 integrin polypeptides are adsorbed to related polypeptidesadhered to insoluble matrix; antibodies specific to human α5β1 integrinpolypeptides will flow through the matrix under the proper bufferconditions. Such screening allows isolation of polyclonal and monoclonalantibodies non-crossreactive to closely related polypeptides(Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988; Current Protocols in Immunology,Cooligan, et al. (eds.), National Institutes of Health, John Wiley andSons, Inc., 1995). Screening and isolation of specific antibodies iswell known in the art (see, Fundamental Immunology, Paul (eds.), RavenPress, 1993; Getzoff et al., Adv. in Immunol. 43: 1-98, 1988; MonoclonalAntibodies: Principles and Practice, Goding, J. W. (eds.), AcademicPress Ltd., 1996; Benjamin et al., Ann. Rev. Immunol. 2: 67-101, 1984).Representative examples of such assays include: concurrentimmunoelectrophoresis, radioimmuno-assay, radioimmuno-precipitation,enzyme-linked immuno-sorbent assay (ELISA), dot blot or Western blotassay, inhibition or competition assay, and sandwich assay. For a reviewof immunological and immunoassay procedures, see Basic and ClinicalImmunology (Stites & Terr eds., 7^(th) ed. 1991).

IV. Methods for Measuring Efficacy in Modulating Angiogenesis

The present invention provides methods for evaluating physiologicaleffects modulated by a humanized anti-α5β1 integrin antibody. As athreshold issue, these methods allow screening of compositionscomprising the antibodies of the present invention to determine safe,effective therapeutic dosages. During treatment, some of these methodsare applicable to monitoring progress, and modulating dosage to provideoptimal clinical effect.

The methods comprise providing a viable tissue that is compatible toanalysis or treatment; i.e., a tissue that when injured, includingimmortalization, undergoes undesirable choroidal neovascularizationevents, which if inhibited or prevented would improve the prognosis ofthe patient and/or healing of the injured tissue. Typical tissuessuitable for treatment or study include tumors and eye tissue,particularly the macula of the eye. The term “tumor” is used broadlyherein to mean any new, pathological tissue growth. For purposes of thepresent invention, a tumor is characterized, in part, by angiogenesis. Atumor can be benign, for example, a hemangioma, glioma, teratoma, andthe like, or can be malignant, for example, a carcinoma, sarcoma,glioblastoma, astrocytoma, neuroblastoma, retinoblastoma, and the like.The term “tumor” is used generally to refer to a benign or malignanttumor, and the term “cancer” is used generally to refer to a malignanttumor, which may or may not be metastatic. Malignant tumors that can bediagnosed using a method of the invention include, for example,carcinomas such as lung cancer, breast cancer, prostate cancer, cervicalcancer, pancreatic cancer, colon cancer and ovarian cancer; and sarcomassuch as osteosarcoma and Kaposi's sarcoma, provided the tumor ischaracterized, at least in part, by angiogenesis associated with α5β1expression by the newly forming blood vessels For study, these tissuescan be isolated by procedures known and sources readily available tothose of skill in the art.

When using a viable tissue for testing the efficacy of the therapeuticantibodies of the present invention, the tissue must first be injured tocreate lesions and promote choroidal neovascularization. Injury may beaccomplished by any suitable means, including mechanical, chemical, orbiological means. Exemplary mechanical means of injury include cutting,piercing or clamping. Chemical means include applying agents to thetissue that cause necrosis, apoptosis, or loss of cell to cell contact.Biological means include treatment with infectious agents, such asviruses, bacteria or prions. A preferred method of creating lesions isthrough the use of a laser. Any laser capable of injuring the tissue maybe used, with CO₂ gas lasers being a preferred type, a most preferredtype being a OCULIGHT® GL (532 nm) Laser Photo-coagulator with a IRISMedical® Portable Slit Lamp Adaptor. Other laser sources are alsosuitable provided they can produce laser light from about 300 to about700 mwatts, and lesions less than 200 μm, preferably less than 100 μm,more preferably from about 50 to about 100 μm in diameter, and mostpreferably about 75 to 25 μm in diameter. Typically the laser light isapplied to the tissue for a fraction of a second. Normally less than 0.5second, more preferably less than 0.1 second, most preferably less than0.05 second.

The antibody applied is a chimeric or humanized anti-α5β1 integrinantibody. Preferably, this antibody comprises a variable heavy chainregion having a sequence 65%, preferably more than 75%, more preferably85%, 90%, 95%, 97% or 99% identical to an amino acid sequence selectedfrom the group consisting of SEQ ID NOS.: 1-6, 16 and 20 and a variablelight chain region independently selected and having a sequence 65%,preferably more than 75%, more preferably 85%, 90%, 95%, 97% or 99%identical to an amino acid sequence from the group consisting of SEQ IDNOS.: 7-12, 18 and 22. Most preferably, the chimeric or humanizedanti-α5β1 integrin antibody comprises a variable heavy chain regionhaving a sequence selected from the group consisting of SEQ ID NOS.:2-6, 16, 20, 25, 28 and 31 and a variable light chain regionindependently selected from the group consisting of SEQ ID NOS.: 8-12,18, 22, 26 and 32.

Antibodies of the present invention can be administered by variousroutes, for example, intravenously, orally, or directly into the regionto be treated, for example, directly into a neoplastic tumor; via eyedrops, where the pathological condition involves the eye; orintrasynovially, where the condition involves a joint.

The amount of therapeutic antibody that is administered to an individualwill depend, in part, on whether the agent is administered for adiagnostic purpose or for a therapeutic purpose. Methods for determiningan effective amount of an agent to administer for a diagnostic or atherapeutic procedure are well known in the art and include phase I,phase II and phase III clinical trials. Using the methods of the presentinvention, effective amounts can be determined by, for example,titrating dosages to individual test subjects and charting progress as afunction of neoangiogenic inhibition.

The total amount of the pharmaceutical that can be administered to asubject as a single dose, either as a bolus or by infusion over arelatively short period of time, or can be administered using afractionated treatment protocol, in which the multiple doses areadministered over a more prolonged period of time. As noted above, oneskilled in the art would know that the concentration of a particularagent required to provide an effective amount to a region or regions ofangiogenesis associated with α5β1 integrin expression in an individualdepends on many factors including the age and general health of thesubject, as well as the route of administration, the number oftreatments to be administered, and the nature of the pharmaceutical. Inview of these factors, the skilled artisan would adjust the particulardose so as to obtain an effective amount for efficaciously interferingwith the specific binding of α5β1 integrin with its ligand, therebyallowing for reducing or inhibiting of angiogenesis.

Monitoring of clinically relevant progress is another aspect of thepresent invention. Monitoring may be carried out by any suitable methodknown in the art. Preferred methods include microscopy, Nuclear MagneticResonance and X-ray. In the case of eye tissue, indirect ophthalmoscopicexamination of the posterior chamber of the eye, and biomicroscopicexamination of the anterior segment of the eye can be used. A preferredmethod of monitoring the extent of choroidal neovascularization is byintravenously a fluorescein dye, and examining the viable tissue byfluorescein angiography.

A preferred method of screening the effectiveness of anti-α5β1 integrinantibodies in inhibiting or preventing neo angiogenesis is by creatinglesions in the retina of an animal, applying anti-α5β1 integrinantibodies to the lesions, and then monitoring the progression ofneoangiogenesis in the damaged tissue relative to suitable controlexperiments. This approach is discussed in detail in Example 6, below.These studies have led to the surprising finding that application ofanti-α5β1 integrin antibodies to one eye of an individual results intreatment of lesions present in both eyes of the individual. It issuggested that newly-formed blood vessels in the injured tissue are“leaky” and results in antibodies applied to one eye entering thesystemic blood, which carries them to the other eye. This result holdsregardless of whether whole antibodies or Fab fragments are used in thetreatment. These results indicate a novel method of treating eye lesionsby administering therapeutic anti-α5β1 integrin antibodies of thepresent invention systemically by, for example, intravenous injection.

V. Therapeutic Uses

An additional embodiment of the invention includes pharmaceuticalcompositions comprising the therapeutic antibodies described herein.These compositions may contain agents that enhance the uptake orlocalization of the therapeutic component, decrease inflammation, orotherwise provide localized relief.

The antibodies of the present invention that are useful in reducing orinhibiting angiogenesis associated with α5β1 integrin expression, or apharmaceutical composition containing the antibodies, can be used fortreating any pathological condition that is characterized, at least inpart, by angiogenesis. One skilled in the art would know that the agentcan be administered by various routes including, for example, orally, orparenterally, including intravenously, intramuscularly, subcutaneously,intraorbitally, intracapsularly, intrasynovially, intraperitoneally,intracisternally or by passive or facilitated absorption through theskin using, for example, a skin patch or transdermal iontophoresis.Furthermore, the antibodies can be administered by injection,intubation, via a suppository, orally or topically, the latter of whichcan be passive, for example, by direct application of an ointment orpowder containing the antibodies, or active, for example, using a nasalspray or inhalant. The antibodies can also be administered as a topicalspray, if desirable, in which case one component of the composition isan appropriate propellant. The pharmaceutical composition also can beincorporated, if desired, into liposomes, microspheres or other polymermatrices (Gregoriadis, Liposome Technology, Vol. 1 (CRC Press, BocaRaton, Fla. 1984), which is incorporated herein by reference).Liposomes, for example, which consist of phospholipids or other lipids,are nontoxic, physiologically acceptable and metabolizable carriers thatare relatively simple to make and administer.

Angiogenesis associated with α5β1 integrin expression can occur locally,for example, in the retina of an individual suffering from diabeticretinopathy, or can occur more systemically, for example, in anindividual suffering from rheumatoid arthritis or a metastatic malignantneoplasm. Since regions of angiogenesis can be localized or can moresystemically dispersed, one skilled in the art would select a particularroute and method of administration of the therapeutic antibodies of thepresent invention based, in part, on this factor.

For example, in an individual suffering from diabetic retinopathy, whereangiogenesis associated with α5β1 integrin expression is localized tothe retina, the agent may be formulated in a pharmaceutical compositionconvenient for use as eye drops, which can be administered directly tothe eye. In comparison, in an individual suffering from a metastaticcarcinoma, the agent in a pharmaceutical composition that can beadministered intravenously, orally or by another method that distributesthe agent systemically. Thus, antibodies of the present invention can beadministered by various routes, for example, intravenously, orally, ordirectly into the region to be treated, for example, directly into aneoplastic tumor; via eye drops, where the pathological conditioninvolves the eye; or intrasynovially, where the condition involves ajoint.

A therapeutic antibody is administered in an effective amount, which isan amount sufficient to interfere with the specific binding of α5β1integrin to its specific ligand in an individual. Generally, an agentantagonist is administered in a dose of about 0.0001 to 100 mg/kg bodyweight, though these will vary somewhat with the application. Based onthe results of efficacy trials discussed above, the artisan would beable to determine an effective dosage range for a given treatment.Estimates of an amount to be administered can be adjusted accordingly,for example, where the agent is to be administered locally.

A preferred method of administering the antibodies of the presentinvention is by way of injection, either intradermally, intravenously ordirectly into the joint or tissue that has suffered an injury. Forexample, when retinal tissue has been damaged, therapeutic antibodies ofthe present invention can be injected intravitreally into an affectedeye. A surprising result of the present invention is that treatmentapplied to one eye leads to clinically beneficial effects in both eyes(assuming both eyes are injured). It appears that newly formed bloodvessels are “leaky,” allowing antibodies applied to the first eye topass into the blood stream where they are transported to the second eye.When applied to the eye in this manner, the dose is preferably less than5 μM, more preferably between 0.5 and 2 μM, and most preferably between0.1 and 1.0 μM. Where indicated, treatment can take the form of multipledoses, given over an area or period of time. Dosage in a multiple formatmay all be identical, or can be independently determined and applied.This result has also led to an additional method of treating lesionswith associated neoangiogenesis comprising systemic application of aneffective amount of a therapeutic antibody (for example by intravenousinjection) wherein neoangiogenesis of an injured tissue is inhibited orprevented.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for clarity and understanding, it willbe readily apparent to one of ordinary skill in the art in light of theteachings of this invention that certain changes and modifications maybe made thereto without departing from the spirit and scope of theappended claims.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES

As can be appreciated from the disclosure provided above, the presentinvention has a wide variety of applications. Accordingly, the followingexamples are offered for illustration purposes and are not intended tobe construed as a limitation on the invention in any way. Those of skillin the art will readily recognize a variety of noncritical parametersthat could be changed or modified to yield essentially similar results.

Example 1 Construction of M200 Chimera from Murine IIA1 Anti-α5β1Integrin

This example describes construction of the chimeric antibody M200.

A. Starting DNA Sequences of IIA1 and 200-4 V_(H) and V_(L) Domains

The variable heavy (V_(H)) and light (V_(L)) domains of the mouseanti-human α5β1 integrin antibody, IIA1 (Pharmingen, San Diego Calif.)were cloned from the IIA1 hybridoma cDNA and sequenced as part of theinitial construction of the 200-4 antibody. FIG. 3 shows the cDNAsequences of the IIA1 V_(H) (SEQ ID NO: 13) and V_(L) (SEQ ID NO: 14)domains. During the construction of the 200-4 mouse/human chimeric IgG4antibody from IIA1, silent XhoI restriction sites (CTCGAG) (SEQ ID NO:33) were introduced into the framework 4 regions of both IIA1 V_(H) andV_(L). The 200-4 V_(H) (SEQ ID NO: 15) and V_(L) (SEQ ID NO: 17) DNAsequences containing these silent XhoI sites, as found in expressionconstructs DEF38 IIA1/human G4 chimera and NEF5 IIA1/K chimera, areshown in FIG. 4. These 200-4 V_(H) and V_(L) sequences were used as thestarting point for all subsequent recombinant DNA manipulations.

B. Design of M200 V_(H) and V_(L) Mini-Exons

The 200-4 V_(H) and V_(L) domains in expression plasmids DEF38IIA1/human G4 chimera and NEF5 IIA1/K chimera are directly fused totheir adjacent constant domains through silent XhoI sites, with nointervening introns. In order to make these variable domains compatiblewith the desired antibody expression vectors based on the genomic DNA,it was necessary to design ‘mini-exons’ which recreate functional donorsplice sites at the 3′ ends of the variable coding region. Sequencecomparisons revealed that the V_(H) and V_(L) regions of IIA1 utilizedthe murine JH4 and JK1 segments, respectively; therefore the mini-exonswere designed to recreate natural murine JH4 and JK1 donor splice sitesfollowing the last amino acid in the V_(H) and V_(L) domains. Inaddition, the XhoI sites were removed, restoring the framework 4sequences as found in the original IIA1 hybridoma. The mini-exons wereflanked with restriction sites: 5′ and 3′ XbaI sites (TCTAGA) (SEQ IDNO: 34) for the V_(H) mini-exon, and 5′ MluI (ACGCGT) (SEQ ID NO: 35)and 3′ XbaI (TCTAGA) (SEQ ID NO: 34) for the V_(L) mini-exon.

Recombinant antibody variable domains occasionally contain undesiredalternative mRNA splice sites, which can then give rise to alternatelyspliced mRNA species. Such sites could, in theory, exist in the murinevariable domain but only become active in the context of a heterogeneousexpression cell and/or new acceptor sites from chimeric constantregions. Taking advantage of codon degeneracy to remove potentialalternative splice sites while leaving the encoded amino acid sequenceunchanged may eliminate such undesired alternative splicing. To detectany potential alternative splice sites in the M200 V_(H) and V_(L)mini-exons, the initial designs were analyzed with a splice siteprediction program from the Center for Biological Sequence Analysis fromthe Technical University of Denmark (www.cbs.dtu.dl/services/NetGene2/).For both 200-M mini-exons, the correct donor splice sites wereidentified; however, potential alternative donor splice sites weredetected in CDR3 of the V_(H) mini-exon and CDR1 of the V_(L) mini-exon.To eliminate the possibility of these splice sites being used, singlesilent base pair changes were made to the mini-exon designs. In the caseof the V_(H) design, a silent GGT to GGA codon change at glycine 100(Kabat numbering) was made; for the V_(L) design, a silent GTA to GTCcodon change at valine 29 was made. In both cases these silent changeseliminated the potential secondary splicing donor signal in the V-genes.

Final designs for the M200 V_(H) and V_(L) mini-exons (SEQ ID NOS: 19,21), containing the flanking restriction sites, murine donor splicesites, with the 200-4 XhoI sites removed, and with the potentialalternative donor splice sites eliminated are shown in FIG. 5.

C. Construction of M200 V_(H) Mini-Exon and Plasmid p200-M-H

The designed mini-exon for M200 V_(H) as shown in FIG. 5A wasconstructed by PCR-based mutagenesis using 200-4 expression plasmidDEF38 IIA1/human G4 chimera as the starting point. Briefly, the 200-4V_(H) region was amplified from DEF38 IIA 1/human G4 chimera usingprimers #110 (5′-TTTTCTAGACCACCATGGCTGTCCTGGGGCTGCTT-3′) (SEQ ID NO:36), which anneals to the 5′ end of the 200-4 V_(H) signal sequence andappends a Kozak sequence and XbaI site, and primer #104(5′-TTTTCTAGAGGTTGTGAGGAC TCACCTGAGGAGACGGTGACTGAGGT-3′) (SEQ ID NO: 37)which anneals to the 3′ end of the 200-4 V_(H) and appends an XbaI site.The 469 bp PCR fragment was cloned into pCR4Blunt-TOPO vector(Invitrogen) and confirmed by DNA sequencing to generate plasmidp200M-V_(H)-2.1. This intermediate plasmid was then used in a second PCRmutagenesis reaction to remove the potential aberrant splice site inCDR3 and to add a murine JH4 donor splice site at the 3′ end of theV_(H) coding region. Two complementary primers, #111(5′-TGGAACTTACTACGGAATGACTA CGACGGGG-3′) (SEQ ID NO: 38) and #112(5′-CCCCGTCGTAGTCATTCCGTAGTAAGTTCCA-3′) (SEQ ID NO: 39) were designed todirect a GGT to GGA codon change at glycine 100 (Kabat numbering) inCDR3 of the M200 V_(H). Primers #110 and #112 were used in a PCRreaction to generate a 395 bp fragment from the 5′ end of the M200 V_(H)mini-exon, and a separate PCR reaction with primers #111 and #113(5′-TTTTCTAGAGGCCATTCTTACCTGAGGAGACGGTGACTGAGGT-3′) (SEQ ID NO: 40)generated a 101 bp fragment from the 3′ end of the M200 V_(H) mini-exon.The two PCR products were gel purified on 1.5% low melting pointagarose, combined, and joined in a final PCR reaction using primers #110and #113. The final 465 bp PCR product was purified, digested with XbaI,and cloned into XbaI-digested and shrimp alkaline phosphatase-treatedvector pHuHCg4.D. The final plasmid, p200-M-H (FIG. 6) was subjected toDNA sequencing to ensure the correct sequence for the 200-M V_(H)mini-exon between the XbaI sites and to verify the correct orientationof the XbaI-XbaI insert.

D. Construction of M200 V_(L) Mini-Exon and Plasmid p200-M-L

The designed mini-exon for M200 V_(L) as shown in FIG. 5B wasconstructed by PCR-based mutagenesis using 200-4 expression plasmid NEF5IIA1/K as the starting point. The V_(L) region was amplified fromNEF5-IIA1-K using primers #101 (5′-TTTACGCGTCCACCATGGATTTTCAGGTGCAGATT-3′) (SEQ ID NO: 41) which anneals to the 5′ endof the signal sequence and appends a Kozak sequence and MluI site, andprimer #102 (5′-TTTTCTAGATTAGGAAAG TGCACTTACGTTTGATTTCCAGCTTGGTGCC-3′)(SEQ ID NO: 42) which anneals to the 3′ end of the 200-4 V_(L) andappends an XbaI site. The 432 bp PCR fragment was cloned intopCR4Blunt-TOPO vector (Invitrogen) and confirmed by DNA sequencing togenerate plasmid p200M-V_(L)-3.3. This intermediate plasmid was thenused in a second PCR mutagenesis reaction to remove the potentialaberrant splice site in CDR1 and to add a murine JK1 donor splice siteat the 3′ end of the V_(L) coding region. Two complementary primers,#114 (5′-TGCCAGTTCAAGTGTCAGTTCCAATTACTTG-3′) (SEQ ID NO: 43) and #115(5′-CAAGTAATTGGAACTGACACTTGA ACTGGCA-3′) (SEQ ID NO: 44) were designedto direct a GTA to GTC codon change at valine 29 (Kabat numbering) inCDR1 of the V_(L) domain. Primers #101 and #115 were used in a PCRreaction to generate a 182 bp fragment from the 5′ end of the V_(L)mini-exon, and a separate PCR reaction with primers #114 and #116(5′-TTTTCTAGACTTTGGATTCTACTTAC GTTTGATTTCCAGCTTGGTGCC-3′) (SEQ ID NO:45) generated a 280 bp fragment from the 3′ end of the V_(L) mini-exon.The two PCR products were gel purified on 1.5% low melting pointagarose, combined, and joined in a final PCR reaction using primers #101and #116. The final 431 bp PCR product was purified, digested with MluIand XbaI, and cloned into MluI- and XbaI-digested light chain expressionvector pHuCkappa.rgpt.dE. The final plasmid, p200-M-L (FIG. 7) wassubjected to DNA sequencing to ensure the correct sequence for the V_(L)mini-exon between the MluI and XbaI sites.

E. Combination of Plasmids p200-M-H and p200-M-L to Make FinalExpression Plasmid p200-M

To express M200 from a single plasmid, p200-M-H and p200-M-L weredigested with EcoRI, and the EcoRI fragment carrying the entire IgG4heavy chain gene from p200-M-H was ligated into EcoRI-linearizedp200-M-L to generate plasmid p200-M (FIG. 8). A large scaleendotoxin-free plasmid preparation of p200-M was prepared from 2.5liters of E. coli culture using the Endotoxin-Free Plasmid Maxi-prep Kit(Qiagen). The plasmid structure was verified by restriction enzymemapping with enzymes BamHI, XbaI, and FspI. The entire coding region forM200 V_(H), V_(L), Cκ and Cγ4 were verified by DNA sequencing. The DNAsequences for the complete M200 heavy (SEQ ID NO: 23) and M200 light(SEQ ID NO: 24) chains are shown in FIG. 9. The corresponding amino acidsequences for the complete M200 heavy (SEQ ID NO: 25) and M200 light(SEQ ID NO: 26) chains are shown in FIG. 10.

Example 2 Generation of Fab Fragment F200 from M200

This example describes making Fab fragment F200.

Fab fragments are generated from M200 IgG starting material by enzymaticdigest. The starting IgG is buffer exchanged into 20 mM sodiumphosphate, 20 mM N-acetyl cysteine pH 7.0. Soluble papain enzyme isadded, and the mixture is rotated at 37° C. for 4 hours. After digestionthe mixture is passed over a protein A column to remove Fc fragments andundigested IgG are removed. Sodium tetrathionate is added to 10 mM andincubated for 30 minutes at room temperature. Finally, this preparationis buffer exchanged into 20 mM sodium phosphate, 100 mM sodium chloride,pH 7.4, to yield the F200 solution.

Because it is a Fab fragment, the F200 light chain DNA and amino acidsequences are the same as the M200 light chain. The complete F200 heavychain DNA (SEQ ID NO: 27) and amino acid (SEQ ID NO: 28) sequences areshown in FIG. 11.

Example 3 In Vitro Inhibition of Endothelial Proliferation by M200

This example describes the effect of the M200 antibody on endothelialproliferation. M200 is a highly specific functional blocking monoclonalantibody against α5β1 integrin.

HUVEC were seeded in 96-well plates at a density of 5000 cells/well inthe presence of various antibodies (M100, M200, anti-VEGF or controlIgG) at the concentrations shown FIG. 14. Plates were pre-treated witheither 10 μg/mL fibronectin or 0.1% poly-L-lysine (PLL) and blocked with2% heat denatured BSA. Cells were grown in defined, serum-free mediumcontaining approximately 2 ng/ml VEGF, bFGF or both. Four days afterplating, total cell viability was assessed by using the tetrazoliumsalt, MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide+++) assay(see e.g. Wasserman & Twentyman, “Use of a colorimetric microtiter (MTT)assay in determining the radiosensitivity of cells from murine solidtumors,” Int J Radiat Oncol Biol Phys. 15(3):699-702 (1988); Romijn, JC, Verkoelen, C F, Schroeder, F H, “Application of the MTT assay tohuman prostate cancer cell lines in vitro: establishment of testconditions and assessment of hormone-stimulated growth and drug-inducedcytostatic and cytotoxic effects,” Prostate 12(1): 99-110 (1988)) Datawere subtracted for background and normalized to a control devoid ofantibody. Each data point was collected in triplicate and the data shownis representative of three individual experiments.

As shown in FIG. 14, HUVEC growth was inhibited by M200 in a dosedependent manner on both PLL and fibronectin (0.40 nM; max inhibition of80%), whereas the control IgG had no effect. Furthermore, M100 (themouse antibody from which M200 was derived) shows an identical abilityto inhibit cell growth.

Importantly, as shown in FIG. 14, the high-affinity, function blockinganti-VEGF mAb, HuMV833 (K_(D)=5.84×10⁻¹¹ nM), exerted significantly lessinhibition of HUVEC growth under all conditions tested (45 nM; maxinhibition of 40%). Challenging the cells with M200 and HuMV833 togetherresulted in no increased inhibitory response.

For the data shown in FIG. 15A, a higher concentration of VEGF (50ng/ml) was included in the HUVEC proliferation assay on fibronectin asdescribed above. As shown in FIG. 15A, HUVEC proliferation onfibronectin stimulated by VEGF is inhibited by M200 to a similar extentas by HuMV833. Thus, M200-mediated cytostatic effects were evident evenat elevated, growth-stimulatory levels of VEGF (50 ng/ml).

Two high affinity antibodies were raised against the M200 idiotyperegion and determined to block binding of M200 to α5β1 integrin. The twoanti-idiotype mAbs (10 μg/ml) were included in the HUVEC proliferationassay described above and assessed for an effect on M200-dependentinhibition of HUVEC growth. Both mAbs are able to inhibit the capacityof a M200 (1 μg/ml) to inhibit HUVEC proliferation. As shown in 15B, theinhibitory activity of M200 was completely reversed by the anti-idiotypemAbs to M200.

Taken together, these results suggest that M200 inhibits HUVECproliferation through a mechanism overlapping that of the anti-VEGFantibody HuMV833 yet also distinct in some aspects.

Example 4 M200 Effect on Endothelial Cell Survival

This example describes the effect of M200 antibody on endothelial cellsurvival.

Antibodies against certain integrins are capable of inducing cell deathin vitro and in vivo. Recently, a function blocking α5β1 mAb was shownto promote apoptosis in cultured human endothelial cells as measured byannexin V staining, caspase-3 cleavage and DNA fragmentation (Kim, etal., 2002).

Similar annexin V staining was carried out on HUVEC grown exposed toM200 or HuMV833. HUVEC grown in serum-free medium (containing VEGF andbFGF, except where indicated) were grown in the presence of M200 (10μg/ml), HuMV833 (10 μg/ml) or staurosporine (5 μM; positive control).Cell death was assessed by staining with Annexin V-alexa488 (green), andHoechst 33258 (blue), followed by fluorescence microscopy (fluorescencemicroscopy images shown in FIG. 16A). In parallel, cell death wasfollowed by flow cytometry 16 hours after plating (FIG. 16B).

As shown in FIGS. 16A and 16B, cells challenged with M200 displayedincreased annexin V staining whereas those challenged with HuMV833 didnot. Thus, M200, in contrast to HuMV833, appears to promote cell deathin endothelial cells.

In addition, the effect of M200 was compared for senescent versusproliferating cells. HUVEC were plated and allowed to proliferate in thepresence of serum and growth factors (middle panel), grown to confluency(left panel) or deprived of serum and growth factors after log phasegrowth (right panel). In each case, cells were left untreated (control)or incubated with M200 (10 μg/ml) or staurosporine (5 μM) for 16 hoursand stained with Annexin V-alexa488.

As shown in FIG. 17, M200 induced cell death in dividing HUVEC, but notHUVEC brought to senescence by either contact inhibition or growthfactor withdrawal. These results suggest that M200 selectively promotescell death in proliferating endothelial cells.

Example 5 Inhibition of In Vitro Tube Formation F200

This example describes a tube formation assay demonstrating in vitroinhibition angiogenesis by F200. HUVECs were mixed as a single cellsuspension into a fibrin clot (prepared from fibrinogen and a-thrombin)together with human serum and a mixture of growth factors (assay in FIG.18A included media supplemented with 0.01 mg/ml rTGF-α and 0.1 mg/ml ofboth VEGF and HGF; assay in FIG. 18B included media supplemented with0.1 mg/ml VEGF alone; and assay in FIG. 18C included media supplementedwith 0.1 mg/ml bFGF alone). Test antibody was added to the media at theindicated concentrations. Over a period of 96 hours, the single cellHUVECs begin to migrate, make contact with other cells and the matrix,form cords and finally 3-dimensional tube-like structures. The extent oftube formation was quantified after 6 days by fixation with 4%formaldehyde and stained with Alexa488˜phalloidin. As shown by theimages and graphs of mean fluorescence depicted in FIG. 18, tubeformation was significantly inhibited by the presence of F200. Tubeformation inhibition was observed in the presence of the growth factors,VEGF, HGF and a mixture of these two with rTGFα.

Example 6 In Vivo Inhibition of CNV in Primate Eyes by M200 and F200

This Example describes the effect of M200 and F200 Fab on vasculardevelopment after laser insult to the maculae of primate eyes.Background literature describing studies of choroidal neovascularizationin animal models include: S. Ryan, “The Development of an ExperimentalModel of Subretinal Neovascularization in Disciform MacularDegeneration,” Transactions of the American Ophthalmological Society 77:707-745 (1979); S. J. Ryan, “Subretinal Neovascularization: NaturalHistory of an Experimental Model,” Archives of Ophthalmology 100:1804-1809 (1982); M. J. Tolentino et al., “Angiography ofFluoresceinated Anti-Vascular Endothelial Growth Factor Antibody andDextrans in Experimental Choroidal Neovascularization,” Archives ofOphthalmology 118: 78-84(2000).

A. Experimental Design

A total of 8 monkeys were assigned to treatment groups as shown in thetable below. Group N Test Article (left eye) Test Article (right eye) 12 Untreated Buffer (50 μl) 2 2 M200 (1 μM; 50 μL) M200 (1 μM; 50 μL) 3 2F200 (1 μM; 50 μL) F200 (1 μM; 50 μL) 4 1 Control (Rituxan 1 μM; 50 μL)M200 (1 μM; 50 μL) 5 1 Control (Rituxan 1 μM; 50 μL) F200 (1 μM; 50 μL)

M200 and F200 were administered in a carrier buffer solution. Rituxanwas used as the control dose. Choroidal neovascularization (CNV) wasinduced on Day 1 by laser treatment to the maculae of both eyes of eachanimal as described below. All animals were dosed with M200, F200, orControl as indicated in the table once weekly for 4 weeks. The first dayof dosing was designated Day 1. The animals were evaluated for changesin clinical signs, body weight, and other parameters, using standardtechniques. All animals were euthanized on Day 28.

B. Laser Induction of Chioroidal Neovascularization (CNV)

The animals were fasted overnight prior to laser treatment and dosing.The animals were sedated with ketamine HCl (intramuscular, to effect)followed by a combination of intravenous ketamine and diazepam (toeffect) for the laser treatment and dosing procedure.

Choroidal neovascularization (CNV) was induced by laser treatment to themaculae of both eyes. Lesions were placed in the macula in a standard9-spot grid pattern with a laser [OCULIGHT® GL (532 nm) LaserPhoto-coagulator with an IRIS Medical® Portable Slit Lamp Adaptor].Laser spots in the right eye mirror placement in the left eye. Theapproximate laser parameters were as follows: spot size: 50-100 μm;laser power: 300-700 milliwatts; exposure time: 0.1 seconds. Parametersfor each animal were recorded on the day of laser treatment. Photographswere taken using a TRC-50EX Retina Camera and/or SL-4ED Slit Lamp, withdigital CCD camera.

C. Dosing

An intravitreal injection of immunoglobulin (test) or control articlewas performed in each eye. Injection on Day 1 occurs immediatelyfollowing laser treatment. Prior to dose administration, a mydriatic (1%tropicamide) was instilled in each eye. Eyes were rinsed with a diluteantiseptic solution (5% Betadine solution or equivalent), the antisepticwas rinsed off with 0.9% sterile saline solution (or equivalent) and twodrops of a topical anesthetic (proparacaine or equivalent) was instilledin the eye. A lid speculum was inserted to keep the lids open during theprocedure and the globe was retracted. The needle of the dose syringewas passed through the sclera and pars plana approximately 4 mmposterior to the limbus. The needle was directed posterior to the lensinto the mid-vitreous. Test article was slowly injected into thevitreous. Forceps were used to grasp the conjunctiva surrounding thesyringe prior to needle withdrawal. The conjunctiva was held with theforceps during and briefly following needle withdrawal. The lid speculumwas then removed. Immediately following dosing, the eyes were examinedwith an indirect ophthalmoscope to identify any visible post-dosingproblems. A topical antibiotic (TOBREX® or equivalent) can be dispensedonto each eye to prevent infection immediately following dosing and oneday after dosing. The animals were returned to their cages whensufficiently recovered from the anesthetic.

Dosing was done on a weekly basis following the schedule in the tablebelow: Number of Test Test Dose Group Animals Article Dose Article DoseVolume No. (M/F) (left eye) Level (right eye) Level (μL/eye) 1 1/1 noneNA Buffer 0 50 2 1/1 M200 300 μg M200 300 μg 50 3 1/1 F200 100 μg F200100 μg 50 4 1/0 Control 100 μg M200 300 μg 50 5 1/0 Control 100 μg F200100 μg 50

The gram amount dose levels indicated were for each eye. Assuming anaverage eye volume of 2 ml, the dose per eye was˜150 μg/ml M200 and 50μg/ml F200. In both cases, the molar concentration of M200 or F200 was 1μM.

D. Monitoring Inhibition of Angiogenesis

Indirect ophthalmoscopy was used to examine the posterior chamber, andbiomicroscopy was used to exam the anterior segment of the eye. The eyeswere scored using standard procedures (Robert B. Hackett and T. O.McDonald. 1996, Dermatotoxicology. 5th Edition. Ed. By E. B. Marzulliand H. I. Maibach. Hemisphere Publishing Corp., Washington, D.C.).

Fluorescein angiography was performed prior to lesion formation and 5,12, 19 and 26 days subsequent to lesions and initial treatment. Acombination of ketamine and diazepam (approximately 10 mg/kg ketamineand 0.5 mg/kg diazepam, intravenously) can be given to maintainsedation. Lid speculums were used to retract the eyelids. Prior toadministration of fluorescein dye, each animal was placed in anophthalmology chair that will maintain the position of the head duringphotography. Photographs were taken, using a fundus camera (TRC-50EXRetina Camera). Images captured using the Topcon IMAGENET™ data capturesystem. Fluorescein dye (10% fluorescein sodium, approximately 0.1mL/kg) was injected via a cephalic or saphenous vein. Color andblack-and-white photographs were taken at several time points followingdye injection, including the arterial phase, early arteriovenous phaseand several late arteriovenous phases in order to monitor leakage offluorescein associated with CNV lesions. The unchanged images can betransferred to compact discs for storage and shipment.

In addition, the eyes may be photographed (TRC-50EX Retina Camera and/orSL-4ED Slit Lamp, with digital CCD camera). The animals may be lightlysedated with ketamine HCl prior to this procedure, and a few drops of amydriatic solution (typically 1% tropicamide) was instilled into eacheye to facilitate the examination.

E. Results

Analysis of fluorescein angiography images generated using these groupsclearly indicates presence of CNV at day 13 and day 20. CNV persisteduntil day 28 in control groups (e.g. Groups 1, 4 (left eye) and 5 (lefteye)). In contrast, the CNV was significantly reduced in M200 andF200-treated eyes (e.g. Groups 2, 3, 4 (right eye) and 5 (right eye)).As shown in FIG. 19, at day 20, an M200 treated eye was showed littleindication of CNV relative to an eye treated only with control.

FIGS. 20-25 show the effect of M200 and F200 on CNV in an individualmonkey's right eye versus the effect of control in the same monkey'sleft eye at days 13, 20 and 27. Significant reduction of CNV isobservable in the individual's eyes treated with either M200 or F200relative to the untreated eyes. The relative reduction in CNV appears tobe greater in the individuals treated with F200. However it is believedthat this apparent difference is due to leakage of the M200 through thebloodstream into the untreated left eye of the individuals. That is,M200 treatment in the individual's right eye also inhibits CNV in theleft eye resulting in less apparent difference between the two eyes. Incontrast, M200 does not leak over to the untreated eye resulting in amuch greater difference in CNV inhibition between the individual's twoeyes.

Example 7 Binding Affinity of M200, F200 and Humanized Variants

A. Kinetic Analyses by Surface Plasmon Resonance

Affinities between AAB1/B2Fc and IIA1, M200 or F200 were analyzed usingBIACORE™ 3000 and 2000 (binding affinity analyzer) (BIAcore, Sweden).IIA1, M200 or F200 was immobilized on the Pioneer F1 chip using standardamine coupling kit (BIAcore). Surface plasmon resonance was measured ata flow rate of 50 ul/min at 24° C. Injection of AAB1/B2Fc (associationphase) occurs over 180 seconds. Dissociation was subsequently monitoredover 3 hours. Kinetics of binding were calculated from data acquired atfive different concentrations of analyte (320 nM, 160 nM, 80 nM, 40 nM,20 nM), using the BIAevaluation program. Double-referencing was appliedto eliminate responses from reference surface and buffer only control.K_(D) was obtained by simultaneously fitting the association anddissociation phases of the sensorgram from the analyte concentrationseries. For M200 K_(D) was determined to be 0.367±0.132 nM. For F200K_(D) was determined to be 0.332±0.065 nM.

B. HuM200 Affinity by Competition ELISA Assay

ELISA binding competition assays may be carried out to determine thebinding affinity of the HuM200 relative to IIA1 and M200.

Wells of 96-well ELISA plates (Nunc-Immuno MaxiSorp plate, NalgeNunc,Naperville, Ill.) were coated with 100 μl of 1.0 μg/ml recombinantsoluble recombinant human α5β1 integrin-Fc fusion protein in 0.2 Msodium carbonate-bicarbonate buffer (pH 9.4) overnight at 4° C. Afterwashing with Wash Buffer (PBS containing 0.1% Tween 20), wells wereblocked with 200 μl of Superblock Blocking Buffer (Pierce) for 30 minand then washed with Wash Buffer. A mixture of biotinylated murine IIA1(0.1 μg/test) and competitor antibody (duplicates of serial 3-folddilutions of competitor antibodies starting at 5 mg/ml) in ELISA Buffer(PBS containing 1% BSA and 0.1% Tween 20) was applied to ELISA plates ina final volume of 100 μl per well. ELISA plates were incubated for 1 hrat room temperature and the wells were washed with Wash Buffer. Then,100 μl of 1/1,000-diluted HRP-conjugated streptavidin (Pierce, Rockford,Ill.) in ELISA Buffer was applied to each well. After incubating for 0.5hr at room temperature and washing with Wash Buffer, 100 μl of TMBsubstrate was added to each well. Absorbance was read at 450 nm using aVERSAmax microplate reader (Molecular Devices, Menlo Park, Calif.).Final competitor concentration in the reaction was plotted versusabsorbance at 450 nm.

HuM200 comprises the heavy and light chain amino acid sequences shown inFIG. 13 (SEQ ID NOS: 31 and 32). HuM200 (also referred to as HuM200-G4)includes a constant region from an IgG4. A second humanized version ofM200, HuM200-g2m3G includes the same variable domains as HuM200 butincludes a constant region of IgG2.

As shown in FIG. 26, the two versions of the humanized M200 antibody,HuM200-G4 and HuM200-g2m3G exhibit a binding affinity curve nearlyidentical to M200. In addition, HuM200-G4 and HuM200-g2m3G have IC₅₀values of 131.8 μg/ml and 102.8 μg/ml, respectively. These values arecomparable to that observed for M200 (106.3 μg/ml), and slightly higherthan IIA1 (79.1 μg/ml).

Example 8 Determination of Efficacy in a Rabbit Model of AdvancedMacular Degeneration (AMD)

Overview

This example illustrates an animal model with an intact retina thatallows for rational development of therapeutic agents against choroidalneovascularization (CNV), which is the hallmark of exudative advancedmacular degeneration (AMD).

A Hydron™ Implant pellet (hydrogel polymer) based sustained-releasesystem for both VEGF and bFGF has been shown to produce floridirreversible retinal NV in the rabbit after intravitreal implantation(See, e.g., Wong et al., “Intravitreal VEGF and bFGF produce floridneovascularization and hemorrhage in the rabbit,” Current Eye Research22: 140-147 (2001)) and to produce CNV following suprachoroidalimplantation (See e.g., Carvalho et al., “Stimulation of choroidalneovascularization in the rabbit through sustained release of VEGF andbFGF,” Poster presentation at “Fifth Annual Vision Research Conference,April 2001” Satellite Symposium of ARVO, Fort Lauderdale Fla.)

In this example, F200 and M200, potent inhibitory antibodies againstα5β1-integrin, are shown to inhibit CNV in this rabbit model as assessedby fundus photograph scoring of degree of hemorrhage, and leakage offluorescein determined by fluorescein angiography (FA). The methodillustrated in this example provides insight into the key targetproteins which, are common downstream mediators from the multitude ofinitial angiogenic signals (such as VEGF) and provides new mechanisticapproaches for the development of therapeutics against exudative AMD.

Surgical Methods and Analysis

In adult male and female Dutch belted rabbits (N=50), a limitedconjunctival peritomy was made in the superotemporal quadrant, followedby a 4 mm full thickness scleral incision concentric to and 3 mmposterior to the limbus. Care was taken not to incise through thechoroid. A Hydron™ Implant pellet (hydrogel polymer) containing 20 μgeach of VEGF and bFGF (Wong et al., “Intravitreal VEGF and bFGF produceflorid neovascularization and hemorrhage in the rabbit,” Current EyeResearch 22: 140-147 (2001)) was placed as posterior as possible to restin the suprachoroidal space, which was created by passing acyclodialysis spatula between the choroid and sclera.

Intravitreal injections of M200 (600 mg) and F200 (200 mg) in citratebuffer were made 2 mm posterior to the limbus with a 30-gauge needle atboth time of implant (day 0) and day 15. Intravenous (I.V.) M200 (10mg/kg) was administered at day 0 and day 15. Fundus photographs, OCT,and fluorescein angiographs (FAs) were taken at 1, 2, 3, 4, and 8 weekslater.

Clinical grading of fundus photographs and FAs were performed by twomasked graders on a scale of 0, 1 (mild), 2 (moderate), 3 (moderatelysevere), and 4 (severe). Generally, increased hemorrhaging as indicatedby areas of deeper and/or darker redness in the fundus photographsresults in increased scores. The clinical grading scores for the imagesare included beside each fundus photograph. Animals were enucleated atweek 4 (N=40) and week 8 (N=10) for histology.

Results

The VEGF/bFGF Hydron™ Implant pellets (hydrogel polymer) produced arobust, persistent model with high penetrance and yielded 75% of rabbitswith CNV. In this robust rabbit model of CNV, 5 of 8 (62.5%) ofimplanted control eyes developed CNV by week 4. FIG. 27 showsrepresentative fundus photographs and accompanying FAs for rabbit eyesfrom the four treatment groups.

Treatment with M200 or F200 resulted in significant inhibition ofsub-retinal hemorrhaging due to the VEGF/bFGF implant. FIG. 28 depictsfour plots of results from analysis of fundus photographs and FAs. Asshown in the top two plots in FIG. 28, clinical grading of the fundusphotographs taken over the course of the treatment period revealedsignificant inhibition of subretinal hemorrhage for treatment groupscompared to placebo. For intravitreal M200, p=0.130, 0.03, 0.003, 0.001for weeks 1-4 respectively. For intravitreal F200, p=0.042, 0.004,0.002, 0 for weeks 1-4. For intravenous M200, p=0.009, 0.001, 0.005, 0for weeks 1-4. As shown in the bottom two plots of FIG. 28, grading ofthe FA images also showed trends toward inhibition of CNV.Interestingly, the parent mAb, M200, showed significant inhibition ofCNV by when administered by I.V. route, but intravitreal M200 was lessefficacious than F200.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for clarity and understanding, it willbe readily apparent to one of ordinary skill in the art in light of theteachings of this invention that certain changes and modifications maybe made thereto without departing from the spirit and scope of theappended claims.

1. A method of treatment of a growth factor associated ocular diseasecomprising applying one or more doses of a chimeric or humanized growthfactor neutralizing antibody to the injured eye tissue.
 2. The method ofclaim 1, wherein the growth factor is VEGF.
 3. The method of claim 1,wherein the antibody is an anti-α5β1 integrin antibody that exhibitssignificant inhibition of subretinal hemorrhage due to a VEGF/bFGFhydrogel polymer implant pellet in rabbit eye.
 4. The method of claim 1,wherein the antibody is M200.
 5. The method of claim 1, wherein theantibody is F200.
 6. The method of claim 1, wherein applying comprisesintravitreal injection.
 7. The method of claim 1, wherein applyingcomprises intravenous injection.
 8. The method of claim 1, whereinapplying comprises injecting the one or more doses of antibody into oneeye, whereby the antibody contacts both eyes.
 9. The method of claim 1,wherein the antibody comprises an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 1, 7, 25, 26, 28, 31 and
 32. 10. Themethod of claim 1, wherein the antibody comprises the amino acidsequences of SEQ ID NOs: 1 and
 7. 11. The method of claim 1, wherein theantibody comprises the amino acid sequences of SEQ ID NOs: 25 and 26.12. The method of claim 1, wherein the antibody comprises the amino acidsequences of SEQ ID NOs: 28 and
 26. 13. The method of claim 1, whereinthe antibody comprises the amino acid sequences of SEQ ID NOs: 31 and32.